Crystalline form of a biphenyl compound

ABSTRACT

The invention provides a crystalline 1,2-ethanedisulfonic acid salt of biphenyl-2-ylcarbamic acid 1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-yl ester or a solvate thereof. This invention also provides pharmaceutical compositions comprising such a salt or prepared using such a salt; processes and intermediates for preparing such a salt; and methods of using such a salt to treat a pulmonary disorder.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/328,386, filed on Dec. 16, 2011; which application is a continuationof U.S. application Ser. No. 12/369,605, filed on Feb. 11, 2009 (nowU.S. Pat. No. 8,101,766 B2); which application is a continuation of U.S.application Ser. No. 11/204,065, filed on Aug. 15, 2005 (now U.S. Pat.No. 7,521,558 B2); which application claims the benefit of U.S.Provisional Application No. 60/601,805, filed on Aug. 16, 2004; theentire disclosures of which are incorporated herein by reference intheir entirety.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to novel crystalline 1,2-ethanedisulfonicacid salts of a biphenyl compound which are expected to be useful astherapeutic agents for treating pulmonary disorders. This invention alsorelates to pharmaceutical compositions comprising such crystallinecompounds or prepared from such crystalline compounds, processes andintermediates for preparing such crystalline compounds and methods ofusing such crystalline compounds to treat a pulmonary disorder.

2. State of the Art

Commonly-assigned U.S. patent application Ser. No. 10/779,157, filed onFeb. 13, 2004, disclose novel biphenyl compounds that are useful astherapeutic agents for treating pulmonary disorders, such as chronicobstructive pulmonary disease (COPD) and asthma. In particular, thecompound, biphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester is specifically disclosed in these applications as possessing bothmuscarinic antagonist and β₂ adrenergic receptor agonist activity. Thechemical structure of biphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester is represented by formula I:

Therapeutic agents useful for treating pulmonary disorders areadvantageously administered directly into the respiratory tract byinhalation. In this regard, several types of pharmaceutical inhalationdevices have been developed for administering therapeutic agents byinhalation including dry powder inhalers (DPI), metered-dose inhalers(MDI) and nebulizer inhalers. When preparing pharmaceutical compositionsand formulations for use in such devices, it is highly desirable to havea crystalline form of the therapeutic agent that is neither hygroscopicnor deliquescent and which has a relatively high melting point (i.e.greater than about 150° C.) thereby allowing the material to bemicronized without significant decomposition or loss of crystallinity.

No crystalline salt forms of the compound of formula I have beenreported previously. Accordingly, a need exists for a stable,non-deliquescent crystalline salt form of the compound of formula Iwhich has an acceptable level of hygroscopicity and a relatively highmelting point.

SUMMARY OF THE INVENTION

The present invention provides crystalline 1,2-ethanedisulfonic acidsalts of biphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester or a solvate thereof.

Surprisingly, such crystalline 1,2-ethanedisulfonic acid salts of thecompound of formula I have been found not to be deliquescent, even whenexposed to atmospheric moisture. Additionally, such crystalline saltshave an acceptable level of hygroscopicity and a very high meltingpoint, e.g., greater than about 215° C. In a particular embodiment, acrystalline salt of the present invention has a melting point greaterthan about 230° C.

Among other uses, a crystalline 1,2-ethanedisulfonic acid salt of thecompound of formula I is useful for preparing pharmaceuticalcompositions which are expected to be useful for treating pulmonarydisorders. Accordingly, in another of its composition aspects, thepresent invention provides a pharmaceutical composition comprising apharmaceutically acceptable carrier and a 1,2-ethanedisulfonic acid saltof biphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl{-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester or a solvate thereof.

In a particular embodiment, the pharmaceutical composition of thisinvention further comprises a steroidal anti-inflammatory agent, such asa cortecosteroid; or a phosphodiesterase-4 inhibitor; or a combinationthereof.

In another embodiment, this invention provides a pharmaceuticalcomposition comprising an aqueous isotonic saline solution comprising a1,2-ethanedisulfonic acid salt of biphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester, wherein the solution has a pH in the range of from about 4 toabout 6.

In yet another embodiment, this invention provides a combinationcomprising:

(a) a crystalline 1,2-ethanedisulfonic acid salt ofbiphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester or a solvate thereof; and

(b) a steroidal anti-inflammatory agent.

The compound of formula I has both muscarinic antagonist and β₂adrenergic receptor agonist activity. Accordingly, a1,2-ethanedisulfonic acid salt of this invention is expected to beuseful as a therapeutic agent for treating pulmonary disorders, such asasthma and chronic obstructive pulmonary disease.

Accordingly, in one of its method aspects, this invention provides amethod for treating a pulmonary disorder, the method comprisingadministering to a patient in need of treatment a therapeuticallyeffective amount of a 1,2-ethanedisulfonic acid salt ofbiphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester or a solvate thereof.

Additionally, in another of its method aspects, this invention providesa method of producing bronchodilation in a patient, the methodcomprising administering by inhalation to the patient abronchodilation-producing amount of a 1,2-ethanedisulfonic acid salt ofbiphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]-piperidin-4-ylester or a solvate thereof.

This invention also provides a method of treating chronic obstructivepulmonary disease or asthma, the method comprising administering to apatient in need of treatment a therapeutically effective amount of a1,2-ethanedisulfonic acid salt of biphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester or a solvate thereof.

This invention is also directed to processes for preparing a crystalline1,2-ethanedisulfonic acid salt of the compound of formula I.Accordingly, in another of its method aspects, this invention provides aprocess for preparing a crystalline 1,2-ethanedisulfonic acid salt ofbiphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester or a solvate thereof; the process comprising contactingbiphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester with 1,2-ethanedisulfonic acid.

In yet another of its method aspects, this invention provides a processfor preparing a crystalline 1,2-ethanedisulfonic acid salt of a compoundof formula I, the process comprising:

(a) contacting a compound of formula II:

wherein R^(1a), R^(1b) and R^(1c) are independently selected from C₁₋₄alkyl, phenyl, —C₁₋₄ alkyl-(phenyl), or one of R^(1a), R^(1b) and R^(1c)is —O—(C₁₋₄alkyl); with fluoride ion; and

(b) contacting the product from step (b) with 1,2-ethanedisulfonic acidor a hydrate thereof; to form a crystalline 1,2-ethanedisulfonic acidsalt of a compound of formula I, wherein step (a) and (b) are conductedin the same reaction vessel without isolation of the product of step(a).

In another of its method aspects, this invention provides a process forpreparing a crystalline 1,2-ethanedisulfonic acid salt of a compound offormula I having a melting point greater than about 230° C., the processcomprising adding a seed crystal of a crystalline 1,2-ethanedisulfonicacid salt of a compound of formula I to a solution comprising a1,2-ethanedisulfonic acid salt of a compound of formula I dissolved inan inert diluent, wherein the seed crystal has a melting point greaterthan about 230° C.

This process can also be used to recrystallize a crystalline1,2-ethanedisulfonic acid salt of a compound of formula I to provide acrystalline form having a melting point greater than about 230° C.Accordingly, the invention further provides a process for preparing acrystalline 1,2-ethanedisulfonic acid salt of a compound of formula Ihaving a melting point greater than about 230° C., the processcomprising:

(a) dissolving a crystalline 1,2-ethanedisulfonic acid salt of acompound of formula I in an inert diluent at a first temperature;

(b) cooling the product of step (a) to a second temperature; and

(c) adding a seed crystal of a 1,2-ethanedisulfonic acid salt of acompound of formula I;

wherein the seed crystal has a melting point higher than about 230° C.,the first temperature is a temperature sufficient to dissolve the1,2-ethanedisulfonic acid salt, and the second temperature is below atemperature at which the seed crystal completely dissolves when added tothe product of step (b).

Additionally, this invention is directed to a process for purifyingbiphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester; the process comprising forming a crystalline 1,2-ethanedisulfonicacid salt of biphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester. This invention is also directed to the products prepared by theprocesses described herein.

This invention is also directed to a crystalline 1,2-ethanedisulfonicacid salt of biphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester or a solvate thereof for use in therapy or as a medicament.

Additionally, this invention is directed to the use of a crystalline1,2-ethanedisulfonic acid salt of biphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester or a solvate thereof for the manufacture of a medicament;especially for the manufacture of a medicament for the treatment of apulmonary disorder.

This invention is also directed to the use of:

(a) a crystalline 1,2-ethanedisulfonic acid salt ofbiphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester or a solvate thereof; and

(b) a steroidal anti-inflammatory agent;

in the manufacture of a medicament for the treatment of a pulmonarydisorder.

This invention is also directed to a crystalline 1,2-ethanedisulfonicacid salt of biphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester or a solvate thereof, in micronized form; and to pharmaceuticalcompositions comprising a pharmaceutically acceptable carrier and acrystalline 1,2-ethanedisulfonic acid salt of biphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]-piperidin-4-ylester or a solvate thereof, in micronized form.

BRIEF DESCRIPTION OF THE DRAWINGS

Various aspects of the present invention are illustrated by reference tothe accompanying drawings.

FIG. 1 shows a differential scanning calorimetry (DSC) trace and athermal gravimetric analysis (TGA) trace and FIG. 2 shows a DSC tracefor samples of a crystalline 1,2-ethanedisulfonic acid salt ofbiphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester of this invention.

FIGS. 3 and 4 show powder x-ray diffraction (PXRD) patterns of samplesof a crystalline 1,2-ethanedisulfonic acid salt of biphenyl-2-ylcarbamicacid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester of this invention.

FIG. 5 shows an infrared (IR) absorption spectra for a crystalline1,2-ethanedisulfonic acid salt of biphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester of this invention.

FIG. 6 shows a dynamic moisture sorption (DMS) trace for a crystalline1,2-ethanedisulfonic acid salt of biphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester of this invention.

DETAILED DESCRIPTION OF THE INVENTION

This invention provides crystalline 1,2-ethanedisulfonic acid salts ofbiphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester or a solvate thereof. The active therapeutic agent in these salts(i.e., the compound of formula I) contains one chiral center having the(R) configuration. However, it will be understood by those skilled inthe art that minor amounts of the (S) stereoisomer may be present in thecompositions of this invention unless otherwise indicated, provided thatany utility of the composition as a whole is not eliminated by thepresence of such an isomer.

The compound of formula l has been named using thecommercially-available AutoNom software (MDL, San Leandro, Calif.).Additionally, 1,2-ethanedisulfonic acid salts are also sometimesreferred to as edisylate salts or edisitate salts.

Definitions

When describing the compounds, compositions, methods and processes ofthis invention, the following terms have the following meanings unlessotherwise indicated.

The term “melting point” as used herein means the temperature at whichthe maximum endothermic heat flow is observed by differential scanningcalorimetry.

The term “micronized form” means a form of particles in which at leastabout 90% of the particles have a diameter of less than about 10 μm.

The term “solvate” means a complex or aggregate formed by one or moremolecules of a solute, i.e. a 1,2-ethanedisulfonic acid salt of thecompound of formula I, and one or more molecules of a solvent. Suchsolvates typically have a substantially fixed molar ratio of solute andsolvent. This term also includes clathrates, including clathrates withwater. Representative solvents include, by way of example, water,methanol, ethanol, isopropanol, acetic acid and the like. When thesolvent is water, the solvate formed is a hydrate.

The term “therapeutically effective amount” means an amount sufficientto effect treatment when administered to a patient in need of treatment.

The term “treating” or “treatment” as used herein means the treating ortreatment of a disease or medical condition (such as COPD) in a patient,such as a mammal (particularly a human) that includes:

-   -   (a) preventing the disease or medical condition from occurring,        i.e., prophylactic treatment of a patient;    -   (b) ameliorating the disease or medical condition, i.e.,        eliminating or causing regression of the disease or medical        condition in a patient;    -   (c) suppressing the disease or medical condition, i.e., slowing        or arresting the development of the disease or medical condition        in a patient; or    -   (d) alleviating the symptoms of the disease or medical condition        in a patient.

The term “unit dosage form” refers to a physically discrete unitsuitable for dosing a patient, i.e., each unit containing apredetermined quantity of a salt of the invention calculated to producethe desired therapeutic effect either alone or in combination with oneor more additional units. For example, such unit dosage forms may be drypowder inhaler capsules, a metered dose from a metered dose inhaler,capsules, tablets, pills, and the like.

1,2-Ethanedisulfonic Acid Salts of the Invention

A crystalline 1,2-ethanedisulfonic acid salt of biphenyl-2-ylcarbamicacid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester of this invention can be prepared from biphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester and 1,2-ethanedisulfonic acid or a hydrate thereof.

A 1,2-ethanedisulfonic acid salt of this invention typically containsbetween about 0.90 and about 1.10 molar equivalents of1,2-ethanedisulfonic acid per molar equivalent of the compound offormula I; including between about 0.95 and about 1.05 molar equivalentsof 1,2-ethanedisulfonic acid per molar equivalent of the compound offormula I. In a particular embodiment, the 1,2-ethanedisulfonic acidsalt of this invention contains about 1 molar equivalent of1,2-ethanedisulfonic acid per molar equivalent of the compound offormula I.

The molar ratio of 1,2-ethanedisulfonic acid to biphenyl-2-ylcarbamicacid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester can be readily determined by various methods available to thoseskilled in the art. For example, such molar ratios can be readilydetermined by ¹H NMR. Alternatively, elemental analysis and HPLC methodscan be used to determine the molar ratio.

The biphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]-piperidin-4-ylester employed in this invention can be readily prepared fromcommercially available starting materials and reagents using theprocedures described in the Examples below; or using the proceduresdescribed in the commonly-assigned U.S. application described in theBackground section of this application.

1,2-Ethanedisulfonic acid is commercially available from, for example,Alfa Chemicals Ltd., Berkshire, UK. In one embodiment, the1,2-ethanedisulfonic acid employed in preparing the salts of thisinvention is a dihydrate. In a particular embodiment, the1,2-ethanedisulfonic acid dihydrate has a purity greater than or equalto 97% (as determined by HPLC). If desired, the 1,2-disulfonic aciddihydrate employed in this invention can be recrystallized from, forexample, acetic acid and acetic anhydride prior to use.

To prepare a crystalline salt of this invention, thebiphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester is typically contacted with about 0.75 to about 1.3 molarequivalents of 1,2-ethanedisulfonic acid or a hydrate thereof.Generally, this reaction is conducted in an inert diluent at atemperature ranging from about 0° C., to about 60° C.; including about20° C. to about 55° C., such as about 25° C. to about 50° C. Suitableinert diluents for this reaction include, but are not limited to,methanol, ethanol, isopropanol, isobutanol, ethyl acetate,dichloromethane and the like optionally containing water. In aparticular embodiment, a solution of 1,2-ethanedisulfonic acid dihydratein ethanol is added to about a five times larger volume ofbiphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester in a mixture of isopropanol and dichloromethane (64:1). In otherparticular embodiments, the solution of 1,2-ethanedisulfonic aciddihydrate includes water or ethanol as the diluent and thebiphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester solution includes isopropanol or ethanol as the diluent.

Alternatively, a crystalline 1,2-ethanedisulfonic acid salt of acompound of formula I can be prepared by contacting a silyl-protectedderivative of the compound of formula I (i.e., a compound of formula II)with a source of fluoride ion and then, in the same reaction vessel,contacting the product with 1,2-ethanedisulfonic acid or a hydratethereof. In a particular embodiment, the silyl-protecting group is atert-butyldimethylsilyl group. Other suitable silyl-protecting groupsinclude tert-butyldiphenylsilyl, diphenylmethylsilyl,di-tert-buytmethylsilyl, tert-butoxydiphenylsilyl and the like. Thesource of fluoride ion used in this process can be any reagentcontaining or comprising fluoride ion or hydrogen fluoride. In aparticular embodiment, the source of fluoride ion is triethylaminetrihydrofluoride. Other suitable sources of fluoride ion includetetrabutylammonium fluoride, potassium fluoride with 18-crown-6,hydrogen fluoride, pyridine hydrofluoride, and the like.

Generally, this process is conducted in an inert diluent at atemperature ranging from about 0° C. to about 50° C.; including about20° C. to about 35° C., such as about 25° C. to about 30° C. Suitableinert diluents for this reaction include, but are not limited to,dichloromethane, methanol and mixtures thereof. In a particularembodiment, a solution of biphenyl-2-ylcarbamic acid1-[2-(4-{[(R)-2-(tert-butyldimethylsilanyloxy)-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-2-chloro-5-methoxy-phenylcarbamoyl)ethyl]piperidin-4-ylester is contacted with about 2.5 to about 3.0 molar equivalents oftriethylamine trihydrofluoride dichloromethane at ambient temperaturefor about 12 to 24 hours or until removal of the silyl group issubstantially complete. To the resulting solution, without isolation ofthe reaction product, is added about 0.9 to about 1.1 molar equivalentsof 1,2-ethanedisulfonic acid dihydrate in methanol and this mixture isheated at about 25° C. to about 35° C. for about 2 to about 6 hours.Upon completion of the reaction, a crystalline 1,2-ethanedisulfonic acidsalt of biphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester is isolated from the reaction mixture by any conventional means,such as precipitation, concentration, centrifugation and the like.

Optionally, a crystalline 1,2-ethanedisulfonic acid salt of thisinvention can be further purified by stirring or slurring the salt inisopropanol containing about 15% to about 25%, including about 20%,water by volume. In a particular embodiment, about 10 mL of theisopropanol/water mixture is employed per gram of the1,2-ethanedisulfonic acid salt.

A process of preparing a crystalline 1,2-ethanedisulfonic acid salt ofthis invention can optionally include the use of a seed crystal toproduce predominately a particular crystalline salt. For example, byusing a seed crystal of a higher melting crystalline salt (i.e., greaterthan about 230° C.), a crystalline 1,2-ethanedisulfonic acid salt of acompound of formula I can be prepared having essentially the samemelting point as the seed crystal. Such seed crystals can be used wheninitially forming the crystalline salt or they can be used torecrystallize a crystalline or partially crystalline salt.

Typically, seed crystals are prepared at small scale by slowcrystallization without stirring and without applying cooling. By way ofillustration, to obtain seed crystals, the crystalline salt is typicallydissolved in an inert diluent at a temperature sufficient to providedissolution. Generally, in the initial process of obtaining seedcrystals, a small quantity, typically less than 10 g, including lessthan 5 g, such as less than 1 g, of the crystalline salt is used. In aparticular embodiment, methanol containing about 12% to about 20% water,including about 13% to about 15% water, is used as the diluent at atemperature ranging from about 60° C. to about 70° C., such as about 60°C. to about 65° C. The solution is allowed to cool to room temperature.After about 1 day to about 3 days, the resulting crystals are isolatedby filtration or other conventional means. Alternatively, seed crystalsmay be obtain from a previous preparation of crystalline material.

In the recrystallization process using seed crystals, a crystalline1,2-ethanedisulfonic acid salt of this invention is dissolved in aninert diluent as in the process of obtaining seed crystals, typicallymethanol containing 15% water, at a temperature ranging from about 60°C. to about 65° C. The solution is allowed to cool to a temperature atwhich the seed crystals do not dissolve, for example to a temperature inthe range of from about 30° C. to about 40° C., and then seed crystalsare added. Typically, the ratio of the weight of seed crystals to theweight of crystalline salt in the solution is between about 1:5 andabout 1:35. The solution is cooled to a temperature at whichcrystallization occurs, for example, to about 20° C. and stirred forabout 2 hours to about 24 hours. The resulting crystals are isolated byconventional means. To obtain sufficient seed crystals to prepare largebatches of material, the recrystallization process can be performedsuccessively using the crystals obtained by a first recrystallization asthe seed crystals for a subsequent recrystallization step. It will beappreciated that the specific temperatures at which the steps of therecrystallization process are performed are selected depending on thecharacter of the diluent and the concentration of the crystalline saltin the diluent. Additionally, the recrystallization process can beconducted using either evaporation or an anti-solvent to facilitatecrystallization instead of cooling.

Among other advantages, it has been discovered that forming acrystalline 1,2-ethanedisulfonic acid salt of the compound of formula Iis useful for purifying the compound of formula I. Generally, acrystalline 1,2-ethanedisulfonic acid salt of this invention has apurity greater than 95%; and typically greater than 98%, as determinedby high performance liquid chromatography.

The crystalline 1,2-ethanedisulfonic acid salt of the present inventionis characterized by a very high melting point as evidenced bydifferential scanning calorimetry (DSC) traces which exhibit a peak inendothermic heat flow in the range of about 215° C. to about 240° C. Ithas been observed that the melting point temperature of the crystallinesalt is dependent on the process by which the crystalline salt wasformed. The seed crystals formed by slow crystallization withoutstirring and without applied cooling exhibit melting points higher thanabout 230° C. Crystalline salts formed by a process includingrecrystallization with such seed crystals typically exhibit meltingpoints in the range of about 230° C. to about 245° C., as shown, forexample, in FIG. 1. Crystalline salts formed without a seed crystalhaving a melting point above about 230° C. typically exhibit meltingpoints in the range of about 215° C. to about 229° C., as shown forexample in FIG. 2. In particular embodiments, therefore, the inventionprovides crystalline 1,2-ethanedisulfonic acid salts of the compound offormula I having a DSC trace in the temperature range above about 200°C. that is substantially in accordance with the trace shown in FIG. 1 orwith that shown in FIG. 2.

In another embodiment, the crystalline 1,2-ethanedisulfonic acid salt ofthe present invention is characterized by a powder x-ray diffraction(PXRD) pattern having significant diffraction peaks at 2θ values of5.0±0.3, and 15.0±0.3. Subtle differences may be observed between thepeak positions in the PXRD spectrum of a crystalline salt prepared byrecrystallization from a high melting point seed crystal, as shown inFIG. 3 and that of a salt prepared without use of such a seed crystal,as shown in FIG. 4. Accordingly in separate embodiments, the crystalline1,2-ethanedisulfonic acid salt of the compound of formula I ischaracterized by a powder x-ray diffraction pattern in which the peakpositions are substantially in accordance with those shown in FIG. 3 orwith those shown in FIG. 4.

In another embodiment, the crystalline 1,2-ethanedisulfonic acid salt ofthe compound of formula I is characterized by its infrared (IR)absorption spectrum which shows significant absorption bands at about704, 748, 768, 841, 900, 1055, 1104, 1166, 1218, 1294, 1408, 1522, 1609,1655, and 1701 cm⁻¹, as illustrated in FIG. 5.

A crystalline 1,2-ethanedisulfonic acid salt of the compound of formulaI has been demonstrated to have a reversible sorption/desorption profilewith an acceptable, moderate level of hygroscopicity (i.e., less thanabout 2.5% weight gain in the humidity range of 40% relative humidity to75% relative humidity).

These properties of the salts of this invention are further illustratedin the Examples below.

Pharmaceutical Compositions and Formulations

The 1,2-ethanedisulfonic acid salt of the compound of formula I istypically administered to a patient in the form of a pharmaceuticalcomposition or formulation. Such pharmaceutical compositions may beadministered to the patient by any acceptable route of administrationincluding, but not limited to, inhaled, oral, nasal, topical (includingtransdermal) and parenteral modes of administration. However, it will beunderstood by those skilled in the art that, once the crystalline saltof this invention has been formulated, it may no longer be incrystalline form, i.e., the salt may be dissolved in a suitable carrier.

Accordingly, in one of its compositions aspects, this invention isdirected to a pharmaceutical composition comprising a pharmaceuticallyacceptable carrier or excipient and a 1,2-ethanedisulfonic acid salt ofbiphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester or a solvate thereof. Optionally, such pharmaceutical compositionsmay contain other therapeutic and/or formulating agents if desired.

The pharmaceutical compositions of this invention typically contain atherapeutically effective amount of a 1,2-ethanedisulfonic acid salt ofbiphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester or a solvate thereof. Typically, such pharmaceutical compositionswill contain from about 0.01 to about 95% by weight of the active agent;including, from about 0.01 to about 30% by weight; such as from about0.01 to about 10% by weight of the active agent.

Any conventional carrier or excipient may be used in the pharmaceuticalcompositions of this invention. The choice of a particular carrier orexcipient, or combinations of carriers or exipients, will depend on themode of administration being used to treat a particular patient or typeof medical condition or disease state. In this regard, the preparationof a suitable pharmaceutical composition for a particular mode ofadministration is well within the scope of those skilled in thepharmaceutical arts. Additionally, the ingredients for such compositionsare commercially available from, for example, Sigma, P.O. Box 14508, St.Louis, Mo. 63178. By way of further illustration, conventionalformulation techniques are described in Remington: The Science andPractice of Pharmacy, 20^(th) Edition, Lippincott Williams & White,Baltimore, Md. (2000); and H. C. Ansel et al., Pharmaceutical DosageForms and Drug Delivery Systems, 7^(th) Edition, Lippincott Williams &White, Baltimore, Md. (1999).

Representative examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, the following: (1)sugars, such as lactose, glucose and sucrose; (2) starches, such as cornstarch and potato starch; (3) cellulose, and its derivatives, such assodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate;(4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8)excipients, such as cocoa butter and suppository waxes; (9) oils, suchas peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil,corn oil and soybean oil; (10) glycols, such as propylene glycol; (11)polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol;(12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions;(21) compressed propellant gases, such as chlorofluorocarbons andhydrofluorocarbons; and (22) other non-toxic compatible substancesemployed in pharmaceutical compositions.

The pharmaceutical compositions of this invention are typically preparedby throughly and intimately mixing or blending a salt of the inventionwith a pharmaceutically acceptable carrier and one or more optionalingredients. If necessary or desired, the resulting uniformly blendedmixture can then be shaped or loaded into tablets, capsules, pills,canisters, cartridges, dispensers and the like using conventionalprocedures and equipment.

In one embodiment, the pharmaceutical compositions of this invention aresuitable for inhaled administration. Suitable pharmaceuticalcompositions for inhaled administration will typically be in the form ofan aerosol or a powder. Such compositions are generally administeredusing well-known delivery devices, such as a nebulizer inhaler, ametered-dose inhaler (MDI), a dry powder inhaler (DPI) or a similardelivery device.

In a specific embodiment of this invention, the pharmaceuticalcomposition comprising the active agent is administered by inhalationusing a nebulizer inhaler. Such nebulizer devices typically produce astream of high velocity air that causes the pharmaceutical compositioncomprising the active agent to spray as a mist that is carried into thepatients respiratory tract. Accordingly, when formulated for use in anebulizer inhaler, the active agent is typically dissolved in a suitablecarrier to form a solution. Suitable nebulizer devices are providedcommercially, for example, by PARI GmbH (Starnberg, German). Othernebulizer devices include Respimat (Boehringer Ingelheim) and thosedisclosed, for example, in U.S. Pat. No. 6,123,068 and WO 97/12687.

A representative pharmaceutical composition for use in a nebulizerinhaler comprises an aqueous solution comprising from about 0.05 μg/mLto about 10 mg/mL of a 1,2-ethanedisulfonic acid salt of compound offormula I or a solvate thereof. In one embodiment, the aqueous nebulizerformulation is isotonic. In one embodiment, the aqueous nebulizerformulation has a pH in the range of from about 4 to about 6. In aparticular embodiment, the aqueous nebulizer formulation is bufferedwith citrate buffer to a pH of about 5. In another particularembodiment, the aqueous nebulizer formulation contains from about 0.1mg/mL to about 1.0 mg/mL free base equivalents of biphenyl-2-ylcarbamicacid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester.

In another specific embodiment of this invention, the pharmaceuticalcomposition comprising the active agent is administered by inhalationusing a dry powder inhaler. Such dry powder inhalers typicallyadminister the active agent as a free-flowing powder that is dispersedin a patient's air-stream during inspiration. In order to achieve a freeflowing powder, the active agent is typically formulated with a suitableexcipient such as lactose, starch, mannitol, dextrose, polylactic acid(PLA), polylactide-co-glycolide (PLGA) or combinations thereof.Typically, the active agent is micronized and combined with a suitablecarrier to form a blend of micronized particles of respirable size,where “micronized particles” or “micronized form” means at least about90% of the particles have a diameter of less than about 10 μm.

A representative pharmaceutical composition for use in a dry powderinhaler comprises lactose having a particle size between about 1 μm andabout 100 μm and micronized particles of a 1,2-ethanedisulfonic acidsalt of compound of formula I, or a solvate thereof.

Such a dry powder formulation can be made, for example, by combining thelactose with the active agent and then dry blending the components.Alternatively, if desired, the active agent can be formulated without anexcipient. The pharmaceutical composition is then typically loaded intoa dry powder dispenser, or into inhalation cartridges or capsules foruse with a dry powder delivery device.

Examples of dry powder inhaler delivery devices include Diskhaler(GlaxoSmithKline, Research Triangle Park, N.C.) (see, e.g., U.S. Pat.No. 5,035,237); Diskus (GlaxoSmithKline) (see, e.g., U.S. Pat. No.6,378,519; Turbuhaler (AstraZeneca, Wilmington, Del.) (see, e.g., U.S.Pat. No. 4,524,769); Rotahaler GlaxoSmithKline) (see, e.g., U.S. Pat.No. 4,353,365) and Handihaler (Boehringer Ingelheim). Further examplesof suitable DPI devices are described in U.S. Pat. Nos. 5,415,162,5,239,993, and 5,715,810 and references cited therein.

In yet another specific embodiment of this invention, the pharmaceuticalcomposition comprising the active agent is administered by inhalationusing a metered-dose inhaler. Such metered-dose inhalers typicallydischarge a measured amount of the active agent or a pharmaceuticallyacceptable salt thereof using compressed propellant gas. Accordingly,pharmaceutical compositions administered using a metered-dose inhalertypically comprise a solution or suspension of the active agent in aliquefied propellant. Any suitable liquefied propellant may be employedincluding chlorofluorocarbons, such as CCl₃F, and hydrofluoroalkanes(HFAs), such as 1,1,1,2-tetrafluoroethane (HFA 134a) and1,1,1,2,3,3,3-heptafluoro-n-propane, (HFA 227). Due to concerns aboutchlorofluorocarbons affecting the ozone layer, formulations containingHFAs are generally preferred. Additional optional components of HFAformulations include co-solvents, such as ethanol or pentane, andsurfactants, such as sorbitan trioleate, oleic acid, lecithin, andglycerin. See, for example, U.S. Pat. No. 5,225,183, EP 0717987 A2, andWO 92/22286.

A representative pharmaceutical composition for use in a metered-doseinhaler comprises from about 0.01% to about 5% by weight of a1,2-ethanedisulfonic acid salt of compound of formula I, or a solvatethereof; from about 0% to about 20% by weight ethanol; and from about 0%to about 5% by weight surfactant; with the remainder being an HFApropellant.

Such compositions are typically prepared by adding chilled orpressurized hydrofluoroalkane to a suitable container containing theactive agent, ethanol (if present) and the surfactant (if present). Toprepare a suspension, the active agent is micronized and then combinedwith the propellant. The formulation is then loaded into an aerosolcanister, which forms a portion of a metered-dose inhaler device.Examples of metered-dose inhaler devices developed specifically for usewith HFA propellants are provided in U.S. Pat. Nos. 6,006,745 and6,143,277. Alternatively, a suspension formulation can be prepared byspray drying a coating of surfactant on micronized particles of theactive agent. See, for example, WO 99/53901 and WO 00/61108.

For additional examples of processes of preparing respirable particles,and formulations and devices suitable for inhalation dosing see U.S.Pat. Nos. 6,268,533, 5,983,956, 5,874,063, and 6,221,398, and WO99/55319 and WO 00/30614.

In another embodiment, the pharmaceutical compositions of this inventionare suitable for oral administration. Suitable pharmaceuticalcompositions for oral administration may be in the form of capsules,tablets, pills, lozenges, cachets, dragees, powders, granules; or as asolution or a suspension in an aqueous or non-aqueous liquid; or as anoil-in-water or water-in-oil liquid emulsion; or as an elixir or syrup;and the like; each containing a predetermined amount of a salt of thepresent invention as an active ingredient.

When intended for oral administration in a solid dosage form (i.e., ascapsules, tablets, pills and the like), the pharmaceutical compositionsof this invention will typically comprise a salt of the presentinvention as the active ingredient and one or more pharmaceuticallyacceptable carriers, such as sodium citrate or dicalcium phosphate.Optionally or alternatively, such solid dosage forms may also comprise:(1) fillers or extenders, such as starches, lactose, sucrose, glucose,mannitol, and/or silicic acid; (2) binders, such ascarboxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone,sucrose and/or acacia; (3) humectants, such as glycerol; (4)disintegrating agents, such as agar-agar, calcium carbonate, potato ortapioca starch, alginic acid, certain silicates, and/or sodiumcarbonate; (5) solution retarding agents, such as paraffin; (6)absorption accelerators, such as quaternary ammonium compounds; (7)wetting agents, such as cetyl alcohol and/or glycerol monostearate; (8)absorbents, such as kaolin and/or bentonite clay; (9) lubricants, suchas talc, calcium stearate, magnesium stearate, solid polyethyleneglycols, sodium lauryl sulfate, and/or mixtures thereof; (10) coloringagents; and (11) buffering agents.

Release agents, wetting agents, coating agents, sweetening, flavoringand perfuming agents, preservatives and antioxidants can also be presentin the pharmaceutical compositions of this invention. Examples ofpharmaceutically acceptable antioxidants include: (1) water-solubleantioxidants, such as ascorbic acid, cysteine hydrochloride, sodiumbisulfate, sodium metabisulfate sodium sulfite and the like; (2)oil-soluble antioxidants, such as ascorbyl palmitate, butylatedhydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propylgallate, alpha-tocopherol, and the like; and (3) metal-chelating agents,such as citric acid, ethylenediamine tetraacetic acid (EDTA), sorbitol,tartaric acid, phosphoric acid, and the like. Coating agents fortablets, capsules, pills and like, include those used for entericcoatings, such as cellulose acetate phthalate (CAP), polyvinyl acetatephthalate (PVAP), hydroxypropyl methylcellulose phthalate, methacrylicacid!methacrylic acid ester copolymers, cellulose acetate trimellitate(CAT), carboxymethyl ethyl cellulose (CMEC), hydroxypropyl methylcellulose acetate succinate (HPMCAS), and the like.

If desired, the pharmaceutical compositions of the present invention mayalso be formulated to provide slow or controlled release of the activeingredient using, by way of example, hydroxypropyl methyl cellulose invarying proportions; or other polymer matrices, such has polylactic acid(PLA) or polylactide-co-glycolide (PLGA), liposomes and/or microspheres.

In addition, the pharmaceutical compositions of the present inventionmay optionally contain opacifying agents and may be formulated so thatthey release the active ingredient only, or preferentially, in a certainportion of the gastrointestinal tract, optionally, in a delayed manner.Examples of embedding compositions which can be used include polymericsubstances and waxes. The active ingredient can also be inmicro-encapsulated form, if appropriate, with one or more of theabove-described excipients.

Suitable liquid dosage forms for oral administration include, by way ofillustration, pharmaceutically acceptable emulsions, microemulsions,solutions, suspensions, syrups and elixirs. Such liquid dosage formstypically comprise the active ingredient and an inert diluent, such as,for example, water or other solvents, solubilizing agents andemulsifiers, such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, oils (esp., cottonseed, groundnut, corn, germ,olive, castor and sesame oils), glycerol, tetrahydrofuryl alcohol,polyethylene glycols and fatty acid esters of sorbitan, and mixturesthereof. Suspensions, in addition to the active ingredient, may containsuspending agents such as, for example, ethoxylated isostearyl alcohols,polyoxyethylene sorbitol and sorbitan esters, microcrystallinecellulose, aluminum metahydroxide, bentonite, agar-agar and tragacanth,and mixtures thereof.

When intended for oral administration, the pharmaceutical compositionsof this invention are preferably packaged in a unit dosage form. Forexample, such unit dosage forms may be capsules, tablets, pills, and thelike.

The salts of this invention can also be administered transdermally usingknown transdermal delivery systems and excipents. For example, acompound of this invention can be admixed with permeation enhancers,such as propylene glycol, polyethylene glycolm monolaurate,azacycloalkan-2-ones and the like, and incorporated into a patch orsimilar delivery system. Additional excipients including gelling agents,emulsifiers and buffers, may be used in such transdermal compositions ifdesired.

The pharmaceutical compositions of this invention may also contain othertherapeutic agents that are co-administered with a 1,2-ethanedisulfonicacid salt of compound of formula I or solvate thereof. For example, thepharmaceutical compositions of this invention may further comprise oneor more therapeutic agents selected from anti-inflammatory agents (e.g.steroidal anti-inflammatory agents, such as corticosteroids; andnon-steroidal anti-inflammatory agents (NSAIDs), phosphodiesterase IVinhibitors, antiinfective agents (e.g. antibiotics or antivirals),antihistamines, β₂ adrenergic receptor agonists, muscarinic receptorantagonistst (i.e., antichlolinergic agents) and the like. The othertherapeutic agents can be used in the form of pharmaceuticallyacceptable salts or solvates. Additionally, if appropriate, the othertherapeutic agents can be used as optically pure stereoisomers.

If desired, the salts of this invention can also be administered incombination with another therapeutic agent or agents, such thosedescribed herein. In this embodiment, the components are not physicallymixed together hut are administered simultaneously or sequentially asseparate compositions. For example, a salt of this invention can beadministered by inhalation simultaneously or sequentially with asteroidal anti-inflammatory agent, such as a corticosteroid, using aninhalation delivery device that employs separate compartments (e.g.blister packs) for each therapeutic agent. Alternatively, thecombination may be administered from multiple delivery devices, i.e.,one delivery device for each therapeutic agent.

Representative β₂ adrenergic receptor agonists that can be used incombination with the compounds of this invention include, but are notlimited to, salmeterol, salbutamol, formoterol, salmefamol, fenoterol,terbutaline, albuterol, isoetharine, metaproterenol, pirbuterol,levalbuterol and the like, or pharmaceutically acceptable salts thereof.Other β₂ adrenergic receptor agonists that can be used in combinationwith the compounds of this invention include, but are not limited to,3-(4-{[6-({(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)phenyl]ethyl}amino)-hexyl]oxy}butyl)benzenesulfonamideand3-(-3-{[7-({(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)phenyl]ethyl}amino)heptyl]oxy}propyl)benzenesulfonamideand related compounds disclosed in WO 02/066422, published on Aug. 29,2002;3-[3-(4-{[6-({(2R)-2-hydroxy-2-[4-hydroxy-3-(hydroxymethyl)phenyl]ethyl}amino)hexyl]-oxy}butyl)phenyl]imidazolidine-2,4-dioneand related compounds disclosed in WO 02/070490, published Sep. 12,2002;3-(4-{[6-({(2R)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)benzenesulfonamide,3-(4-{[6-({(2S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)-benzenesulfonamide,3-(4-{[6-({(2R/S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}benzenesulfonamide,N-(tert-butyl)-3-(4-{[6-({(2R)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]-oxy}butyl)benzenesulfonamide,N-(tert-butyl)-3-(4-{[6-({(2S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)benzenesulfonamide,N-(tert-butyl)-3-(4-{[6-({(2R/S)-2-[3-(formylamino)-4-hydroxyphenyl]-2-hydroxyethyl}amino)hexyl]oxy}butyl)benzenesulfonamideand related compounds disclosed in WO 02/076933, published on Oct. 3,2002;4-{(1R)-2-[(6-{2-[(2,6-dichlorobenzyl)oxy]ethoxy}hexyl)amino]-1-hydroxyethyl}-2-(hydroxymethyl)phenoland related compounds disclosed in WO 03/024439, published on Mar. 27,2003;N-{2-[4-((R)-2-hydroxy-2-phenylethylamino)phenyl]ethyl}-(R)-2-hydroxy-2-(3-formamido-4-hydroxyphenyl)ethylamineand related compounds disclosed in U.S. Pat. No. 6,576,793 B1, issued onJun. 10, 2003;N-{2-[4-(3-phenyl-4-methoxyphenyl)aminophenyl]ethyl}-(R)-2-hydroxy-2-(8-hydroxy-2(1H)-quinolinon-5-yl)ethylamineand related compounds disclosed in U.S. Pat. No. 6,653,323 B2, issued onNov. 25, 2003; and pharmaceutically acceptable salts thereof. In aparticular embodiment, the β₂-adrenoreceptor agonist is a crystallinemonohydrochloride salt ofN-{2-[4-((R)-2-hydroxy-2-phenylethylamino)phenyl]ethyl}-(R)-2-hydroxy-2-(3-formamido-4-hydroxyphenyl)ethylamine.When employed, the β₂-adrenoreceptor agonist will be present in thepharmaceutical composition in a therapeutically effective amount.Typically, the β₂-adrenoreceptor agonist will be present in an amountsufficient to provide from about 0.05 μg to about 500 μg per dose.

Representative steroidal anti-inflammatory agents that can be used incombination with the compounds of this invention include, but are notlimited to, methyl prednisolone, prednisolone, dexamethasone,fluticasone propionate,6,9-difluoro-17-[(2-furanylcarbonyl)oxy]-11-hydroxy-16-methyl-3-oxoandrosta-1,4-diene-17-carbothioicacid S-fluoromethyl ester,6,9-difluoro-11-hydroxy-16-methyl-3-oxo-17-propionyloxy-androsta-1,4-diene-17-carbothioicacid S-(2-oxotetrahydrofuran-3S-yl)ester, beclomethasone esters (e.g.the 17-propionate ester or the 17,21-dipropionate ester), budesonide,flunisolide, mometasone esters (e.g. the furoate ester), triamcinoloneacetonide, rofleponide, ciclesonide, butixocort propionate, RPR-106541,ST-126 and the like, or pharmaceutically-acceptable salts thereof. In aparticular embodiment, the steroidal anti-inflammatory agent is6α,9α-difluoro-17α-[(2-furanylcarbonyl)oxy]-11β-hydroxy-16α-methyl-3-oxoandrosta-1,4-diene-17β-carbothioicacid S-fluoromethyl ester or a pharmaceutically acceptable salt orsolvate thereof. When employed, the steroidal anti-inflammatory agentwill be present in the pharmaceutical composition in a theraputicallyeffective amount. Typically, the steroidal anti-inflammatory agent willbe present in an amount sufficient to provide from about 0.05 μg toabout 500 μg per dose.

Other suitable combinations include, for example, otheranti-inflammatory agents, e.g., NSAIDs (such as sodium cromoglycate;nedocromil sodium; phosphodiesterase (PDE) inhibitors (e.g.theophylline, PDE4 inhibitors or mixed PDE3/PDE4 inhibitors);leukotriene antagonists (e.g. monteleukast); inhibitors of leukotrienesynthesis; iNOS inhibitors; protease inhibitors, such as tryptase andelastase inhibitors; beta-2 integrin antagonists and adenosine receptoragonists or antagonists (e.g. adenosine 2a agonists); cytokineantagonists (e.g. chemokine antagonists such as, an interteukin antibody(IL antibody), specifically, an IL-4 therapy, an IL-13 therapy, or acombination thereof); or inhibitors of cytokine synthesis.

For example, representative phosphodiesterase-4 (PDE4) inhibitors ormixed PDE3/PDE4 inhibitors that can be used in combination with thecompounds of this invention include, but are not limited to cis4-cyano-4-(3-cyclopentyloxy-4-methoxyphenyl)cyclohexan-1-carboxylicacid,2-carbomethoxy-4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-one;cis-[4-cyano-4-(3-cyclopropylmethoxy-4-difluoromethoxyphenyl)cyclohexan-1-ol];cis-4-cyano-4-[3-(cyclopentyloxy)-4-methoxyphenyl]cyclohexane-1-carboxylicacid and the like, or pharmaceutically acceptable salts thereof. Otherrepresentative PDE4 or mixed PDE4/PDE3 inhibitors include AWD-12-281(elbion); NCS-613 (INSERM); D-4418 (Chiroscience and Schering-Plough);CI-1018 or PD-168787 (Pfizer); benzodioxole compounds disclosed inWO99/16766 (Kyowa Hakko); K-34 (Kyowa Hakko); V-11294A (Napp);roflumilast (Byk-Gulden); pthalazinone compounds disclosed in WO99/47505(Byk-Gulden); Pumafentrine (Byk-Gulden, now Altana); arofyliine(Almirall-Prodesarma); VM554/UM565 (Vernalis); T-440 (Tanabe Seiyaku);and T2585 (Tanabe Seiyaku).

Representative muscarinic antagonists (i.e., anticholinergic agents)that can be used in combination with, and in addition to, the compoundsof this invention include, but are not limited to, atropine, atropinesulfate, atropine oxide, methylatropine nitrate, homatropinehydrobromide, hyoscyamine (d,l) hydrobromide, scopolamine hydrobromide,ipratropium bromide, oxitropium bromide, tiotropium bromide,methanthetine, propanthetine bromide, anisotropine methyl bromide,clidinium bromide, copyrrolate (Robinul), isopropamide iodide,mepenzolate bromide, tridihexethyl chloride (Pathilone), hexocycliummethylsulfate, cyclopentolate hydrochloride, tropicamide,trihexyphenidyl hydrochloride, pirenzepine, telenzepine, AF-DX 116 andmethoctramine and the like, or a pharmaceutically acceptable saltthereof; or, for those compounds listed as a salt, alternatepharmaceutically acceptable salt thereof.

Representative antihistamines (i.e., H₁-receptor antagonists) that canbe used in combination with the compounds of this invention include, butare not limited to, ethanolamines, such as carbinoxamine maleate,clemastine fumarate, diphenylhydramine hydrochloride and dimenhydrinate;ethylenediamines, such as pyrilamine amleate, tripelennaminehydrochloride and tripelennamine citrate; alkylamines, such aschlorpheniramine and acrivastine; piperazines, such as hydroxyzinehydrochloride, hydroxyzine pamoate, cyclizine hydrochloride, cyclizinelactate, meclizine hydrochloride and cetirizine hydrochloride;piperidines, such as astemizole, levocabastine hydrochloride, loratadineor its descarboethoxy analogue, terfenadine and fexofenadinehydrochloride; azelastine hydrochloride; and the like, or apharmaceutically acceptable salt thereof; or, for those compounds listedas a salt, alternate pharmaceutically acceptable salt thereof.

Suitable doses for the other therapeutic agents administered incombination with a compound of the invention are in the range of about0.05 mg/day to about 100 mg/day.

The following formulations illustrate representative pharmaceuticalcompositions of the present invention:

FORMULATION EXAMPLE A

A dry powder for administration by inhalation is prepared as follows:

Ingredients Amount Salt of the invention 0.2 mg Lactose  25 mg

Representative Procedure: The compound of the invention is micronizedand then blended with lactose. This blended mixture is then loaded intoa gelatin inhalation cartridge. The contents of the cartridge areadministered using a powder inhaler.

FORMULATION EXAMPLE B

A dry powder formulation for use in a dry powder inhalation device isprepared as follows:

Representative Procedure: A pharmaceutical composition is preparedhaving a hulk formulation ratio of micronized salt of the invention tolactose of 1:200. The composition is packed into a powder inhalationdevice capable of delivering between about 10 μg and about 100 μg of thecompound of the invention per dose.

FORMULATION EXAMPLE C

A dry powder for administration by inhalation in a metered dose inhaleris prepared as follows:

Representative Procedure: A suspension containing 5 wt. % of a salt ofthe invention and 0.1 wt. % lecithin is prepared by dispersing 10 g ofthe compound of the invention as micronized particles with mean sizeless than 10 μm in a solution formed from 0.2 g of lecithin dissolved in200 mL of demineralized water. The suspension is spray dried and theresulting material is micronized to particles having a mean diameterless than 1.5 μm. The particles are loaded into cartridges withpressurized 1,1,1,2-tetrafluoroethane.

FORMULATION EXAMPLE D

A pharmaceutical composition for use in a metered dose inhaler isprepared as follows:

Representative Procedure: A suspension containing 5% salt of theinvention, 0.5% lecithin, and 0.5 % trehalose is prepared by dispersing5 g of active ingredient as micronized particles with mean size lessthan 10 m in a colloidal solution formed from 0.5 g of trehalose and 0.5g of lecithin dissolved in 100 mL demineralized water. The suspension isspray dried and the resulting material is micronized to particles havinga mean diameter less than 1.5 μm. The particles are loaded intocanisters with pressurized 1,1,1,2-tetrafluoroethane.

FORMULATION EXAMPLE E

A pharmaceutical composition for use in a nebulizer inhaler is preparedas follows:

Representative Procedure: An aqueous aerosol formulation for use in anebulizer is prepared by dissolving 0.5 mg of the salt of the inventionin 1 mL of a 0.9% sodium chloride solution acidified with citric acid.The mixture is stirred and sonicated until the active ingredient isdissolved. The pH of the solution is adjusted to a value of about 5 bythe slow addition of NaOH.

FORMULATION EXAMPLE F

Hard gelatin capsules for oral administration are prepared as follows:

Ingredients Amount Salt of the invention 250 mg Lactose (spray-dried)200 mg Magnesium stearate  10 mg

Representative Procedure: The ingredients are throughly blended and thenloaded into a hard gelatine capsule (460 mg of composition per capsule).

FORMULATION EXAMPLE G

A suspension for oral administration is prepared as follows:

Ingredients Amount Salt of the invention 1.0 g Fumaric acid 0.5 g Sodiumchloride 2.0 g Methyl paraben 0.15 g Propyl paraben 0.05 g Granulatedsugar 25.5 g Sorbitol (70% solution) 12.85 g Veegum k (Vanderbilt Co.)1.0 g Flavoring 0.035 mL Colorings 0.5 mg Distilled water q.s. to 100 mL

Representative Procedure: The ingredients are mixed to form a suspensioncontaining 100 mg of active ingredient per 10 mL of suspension.

FORMULATION EXAMPLE H

An injectable formulation is prepared as follows:

Ingredients Amount Salt of the invention 0.2 g Sodium acetate buffersolution (0.4M) 2.0 mL HCl (0.5N) or NaOH (0.5N) q.s. to pH 4 Water(distilled, sterile) q.s. to 20 mL

Representative Procedure: The above ingredients are blended and the pHis adjusted to 4±0.5 using 0.5 N HCl or 0.5 N NaOH.

Utility

The compound of formula I possesses both β₂ adrenergic receptor agonistand muscarinic receptor antagonist activity and therefore, a1,2-ethanedisulfonic acid salt of the compound of formula I of thepresent invention is expected to be useful as a therapeutic agent fortreating medical conditions mediated by β₂ adrenergic receptors ormuscarinic receptors, i.e., medical conditions that are ameliorated bytreatment with a β₂ adrenergic receptor agonist or a muscarinic receptorantagonist. Such medical conditions include, by way of example,pulmonary disorders or diseases including those associated withreversible airway obstruction, such as chronic Obstructive pulmonarydisease (e.g., chronic and wheezy bronchitis and emphysema), asthma,pulmonary fibrosis, allergic rhinitis, rhinorrhea, and the like. Otherconditions which may be treated include premature labor, depression,congestive heart failure, skin diseases (e.g., inflammatory, allergic,psoriatic and proliferative skin diseases, conditions where loweringpeptic acidity is desirable (e.g., peptic and gastric ulceration) andmuscle wasting disease.

Accordingly, in one embodiment, this invention is directed to a methodfor treating a pulmonary disorder, the method comprising administeringto a patient in need of treatment a therapeutically effective amount ofa 1,2-ethanedisulfonic acid salt of biphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester or a solvate thereof. When used to treat a pulmonary disorder, thesalt of this invention will typically be administered by inhalation inmultiple doses per day, in a single daily dose or a single weekly dose.Generally, the dose for treating a pulmonary disorder will range fromabout 10 μg/day to about 200 μg/day.

When administered by inhalation, the compounds of this inventiontypically have the effect of providing bronchodilation. Accordingly, inanother of its method aspects, this invention is directed to a method ofproviding bronchodilation in a patient in need of bronchodilation, themethod comprising administering to the patient abronchodilation-producing amount of a 1,2-ethanedisulfonic acid salt ofbiphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester or a solvate thereof. Generally, the dose for providingbronchodilation will range from about 10 μg/day to about 200 μg/day.

In one embodiment, this invention is directed to a method of treatingchronic obstructive pulmonary disease or asthma, the method comprisingadministering to a patient in need of treatment a therapeuticallyeffective amount of a 1,2-ethanedisulfonic acid salt ofbiphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]-piperidin-4-ylester or a solvate thereof. When used to treat a COPD or asthma, thesalt of this invention will typically be administered by inhalationmultiple doses per day or in a single daily dose. Generally, the dosefor treating COPD or asthma will range from about 10 μg/day to about 200μg/day. As used herein, COPD includes chronic obstructive bronchitis andemphysema (see, for example, Barnes, Chronic Obstructive PulmonaryDisease, N Engl J Med 2000: 343:269-78).

When used to treat a pulmonary disorder, the salt of this invention isoptionally administered in combination with other therapeutic agents.Accordingly, in a particular embodiment, the pharmaceutical compositionsand methods of this invention further comprise a therapeuticallyeffective amount of a steroidal anti-inflammatory agent. The propertiesand utility of 1,2-ethanedisulfonic acid salts of this invention can bedemonstrated using various in vitro and in vivo assays well-known tothose skilled in the art. For example, representative assays aredescribed in further detail in the following Examples.

EXAMPLES

The following Preparations and Examples are provided to illustratespecific embodiments of this invention. These specific embodiments,however, are not intended to limit the scope of this invention in anyway unless specifically indicated.

The following abbreviations have the following meanings unless otherwiseindicated and any other abbreviations used herein and not defined havetheir standard meaning:

-   -   AC adenylyl cyclase    -   Ach acetylcholine    -   ATCC American Type Culture Collection    -   BSA bovine serum albumin    -   cAMP cyclic adenosine monophosphate    -   CHO Chinese hamster ovary    -   cM₅ cloned chimpanzee M₅ receptor    -   DCM dichloromethane (i.e., methylene chloride)    -   DIPEA N,N-diisopropylethylamine    -   dPBS Dulbecco's phosphate buffered saline    -   DMEM Dulbecco's Modified Eagle's Medium    -   DMSO dimethyl sulfoxide    -   EDTA ethylenediaminetetraacetic acid    -   Emax maximal efficacy    -   EtOAc ethyl acetate    -   EtOH ethanol    -   FBS fetal bovine serum    -   FLIPR fluorometric imaging plate reader    -   Gly glycine    -   HATU O-(7-azabenzotriazol-1-yl-N,N,N′,N′-tetramethyluronium        hexafluorophosphate    -   HBSS Hank's buffered salt solution    -   HEK human embryonic kidney cells    -   HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid    -   hM₁ cloned human M₁ receptor    -   hM₂ cloned human M₂ receptor    -   hM₃ cloned human M₃ receptor    -   hM₄ cloned human M₄ receptor    -   hM₅ cloned human M₅ receptor    -   HPLC high-performance liquid chromatography    -   IBMX 3-isobutyl-1-methylxanthine    -   % Eff % efficacy    -   PBS phosphate buffered saline    -   PyBOP benzotriazol-1-yloxytripyrrolidinophosphonium        hexafluorophosphate    -   rpm rotations per minute    -   TFA trifluoroacetic acid    -   THF tetrahydrofuran    -   Tris tris(hydroxymethyl)aminomethane

Unless noted otherwise, reagents, starting materials and solvents werepurchased from commercial suppliers (such as Aldrich, Fluka, Sigma andthe like) and were used without further purification.

In the examples described below, HPLC analysis was conducted using anAgilent (Palo Alto, Calif.) Series 1100 instrument with Zorbax Bonus RP2.1×50 mm columns, supplied by Agilent, (a C14 column), having a 3.5micron particle size. Detection was by UV absorbance at 214 nm. HPLC10-70 data was obtained with a flow rate of 0.5 mL/minute of 10%-70% Bover 6 minutes, Mobile phase A was 2%-98%-0.1% ACN-H₂O-TFA; and mobilephase B was 90%-10%-0.1% ACN-H₂O-TFA. Using the mobile phases A and Bdescribed above, HPLC 5-35 data and HPLC 10-90 data were obtained with a5 minute gradient.

Liquid chromatography mass spectrometry (LCMS) data were obtained withan Applied Biosystems (Foster City, Calif.) model API-150EX instrument.LCMS 10-90 data was obtained with a 10%-90% mobile phase B over a 5minute gradient.

Small scale purification was conducted using an API 150EX PrepWorkstation system from Applied Biosystems. The mobile phase was A:water+0.05% v/v TFA; and B: acetonitrile+0.05% v/v TFA. For arrays(typically about 3 to 50 mg recovered sample size) the followingconditions were used: 20 mL/min flow rate; 15 min gradients and a 20mm×50 mm Prism RP column with 5 micron particles (ThermoHypersil-Keystone, Bellefonte, Pa.). For larger scale purifications(typically greater than 100 mg crude sample), the following conditionswere used: 60 mL/min flow rate; 30 min gradients and a 41.4 mm×250 mmMicrosorb BDS column with 10 micron particles (Varian, Palo Alto,Calif.).

The specific rotation for chiral compounds (indicated as [α]²⁰ _(D)) wasmeasured using a Jasco Polarimeter (Model P-1010) with a tungstenhalogen light source and a 589 nm filter at 20° C. Samples of testcompounds were typically measured at 1 mg/mL water.

Preparation 1 Methyl 4-Amino-5-chloro-2-methoxybenzoate

To a solution of 4-amino-5-chloro-2-methoxybenzoic acid (1.008 g, 5.0mmol) in a mixture of toluene (9 mL) and methanol (1 mL) at 0° C. wasadded (trimethylsilyl)diazomethane (2.0 M in hexane, 3.0 mL, 6.0 mmol)dropwise. The reaction mixture was then warmed to room temperature andstirred for 16 h. Excess (trimethylsilyl)diazomethane was quenched byadding acetic acid until the bright yellow color of the reaction mixturedisappeared. The mixture was then concentrated in vacuo to give thetitle compound as an off-white solid, which was used without furtherpurification.

Preparation 2 Methyl 4-Acryloylamino-5-chloro-2-methoxybenzoate

To crude product of Preparation 2 was added dichloromethane (10 mL, 0.5M) and triethylamine (2.1 mL, 15 mmol). This mixture was cooled to 0° C.and acryloyl chloride (812 μL, 1 0 mmol) was added dropwise withstirring. After 2 h, the reaction was quenched by adding methanol (about2 mL) at 0° C. and the resulting mixture was stirred at room temperaturefor 15 min and then concentrated in vacuo. Dichloromethane (30 mL) andwater (30 mL) were added to the residue and this mixture was mixedthoroughly. The layers were separated and the aqueous layer wasextracted with dichloromethane (20 mL). The organic layers werecombined, dried (Na₂SO₄), filtered and the solvent was removed in vacuoto give the title compound as a brown foamy solid, which was usedwithout further purification.

Preparation 3 Biphenyl-2-ylcarbamic Acid Piperidin-4-yl Ester

Biphenyl-2-isocyanate (97.5 g, 521 mmol) and4-hydroxy-1-benzylpiperidine (105 g, 549 mmol), bothcommercially-available from Aldrich, Milwaukee, Wis., were heatedtogether at 70° C. for 12 h, during which time the formation ofbiphenyl-2-ylcarbamic acid 1-benzylpiperidin-4-yl ester was monitored byLCMS. The reaction mixture was then cooled to 50° C. and ethanol (1 L)was added, and then 6M hydrochloric acid (191 mL) was added slowly. Thereaction mixture was then cooled to ambient temperature and ammoniumformate (98.5 g, 1.56 mol) was added and nitrogen gas was bubbledthrough the solution vigorously for 20 min. Palladium (10 wt. % (drybasis) on activated carbon) (20 g) was then added. The reaction mixturewas heated at 40° C. for 12 h and then filtered through a pad of Celite.The solvent was then removed under reduced pressure and 1M hydrochloricacid (40 mL) was added to the crude residue. Sodium hydroxide (10N) wasthen added to adjust the pH to 12. The aqueous layer was extracted withethyl acetate (2×150 mL) and dried (magnesium sulfate), and then thesolvent was removed under reduced pressure to give the title compound(155 g, 100%). HPLC (10-70) R_(f)=2.52; MS m/z: [M+H⁺] calc'd forC₁₈H₂₀N₂O₂ 297.15; found 297.3.

Preparation 4 Methyl4-{3-[4-(Biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionylamino}-5-chloro-2-methoxybenzoate

To the crude product from Preparation 2 was added the product ofPreparation 3 (1.33 g, 4.5 mmol) and a mixture of THF (22.5 mL) andmethanol (2.5 mL). This mixture was heated at 50° C. with stirring for16 h and then the solvent was removed in vacuo. The residue waschromatographed (silica gel; EtOAc) to give the title compound (0.82 g;R_(f)=0.4, 29% yield over 3 steps) as an off-white foamy solid. MS m/z566.4 (M+H, expected 565.20 for C₃₀H₃₂ClN₃O₆).

Preparation 5 Biphenyl-2-ylcarbamic Acid1-[2-(2-Chloro-4-hydroxymethyl-5-methoxy-phenylcarbamoyl)ethyl]piperidin-4-ylEster

To a solution of the product of Preparation 4 (0.82 mg, 1.45 mmol) in amixture of THF (4.5 mL) and methanol (0.5 mL) at 0° C. was added lithiumborohydride (32 mg, 1.45 mmol). The reaction mixture was allowed to warmto room temperature and was stirred for 41 h. The reaction was thenquenched by adding 1N aqueous hydrochloric acid at 0° C. until no morebubbling was observed and this mixture was stirred for 10 min. Thesolvent was removed in vacuo and the residue was dissolved inacetonitrile (about 2 mL). This solution was purified by prep-RP-HPLC(gradient: 2 to 50% acetonitrile in water with 0.05% TFA). Theappropriate fractions were collected and combined and lyophilized togive the title compound as a trifluoroacetate salt. This salt wastreated with isopropyl acetate (10 mL) and 1N aqueous sodium hydroxide(10 mL) and the organic layer was collected, dried (Na₂SO₄), filteredand the solvent was removed in vacuo to give the title compound (161 mg,21% yield) as a white foamy solid. MS m/z 538.4 (M+H, expected 537.20for C₂₉H₃₂ClN₃O₅).

Preparation 6 Biphenyl-2-ylcarbamic Acid1-[2-(2-Chloro-4-formyl-5-methoxyphenyl-carbamoyl)ethyl]piperidin-4-ylEster

To a solution of the product of Preparation 5 (161 mg, 0.3 mmol) indichloromethane (3 mL) was added dimethyl sulfoxide (213 μL, 3.0 mmol)and diisopropylethylamine (261 μL, 1.5 mmol). This mixture was cooled to−20° C. and sulfur trioxide pyridine complex (238 mg, 1.5 mmol) wasadded slowly. After 30 min, the reaction mixture was quenched by addingwater (about 3 mL). The layers were separated and the organic layer wasdried (Na₂SO₄), filtered and the solvent was removed in vacuo to givethe title compound as a light yellow solid. MS m/z 536.3 (M+H, expected535.19 for C₂₉H₃₀ClN₃O₅).

Preparation 7 8-Benzyloxy-5-(2-bromoacetyl)-1H-quinolin-2-one (a)8-Acetoxy-1H-quinolin-2-one

8-Hydroxyquinoline-N-oxide (160.0 g, 1.0 mol), commercially-availablefrom Aldrich, Milwaukee, Wis., and acetic anhydride (800 mL, 8.4 mol)were heated at 100° C. for 3 h and then cooled in ice. The product wascollected on a Buchner funnel, washed. with acetic anhydride (2×100 mL)and dried under reduced pressure to give 8-acetoxy-1H-quinolin-2-one(144 g) as a solid.

(b) 5-Acetyl-8-hydroxy-1H-quinolin-2-one

A slurry of aluminum chloride (85.7 g, 640 mmol) in 1,2-dichloroethane(280 mL) was cooled in ice, and the product from step (a) (56.8 g, 280mmol) was added. The mixture was warmed to room temperature and thenheated at 85° C. After 30 min, acetyl chloride (1.5 mL, 21 mmol) wasadded and the mixture was heated an additional 60 min. The reactionmixture was then cooled and added to 1N hydrochloric acid (3 L) at 0° C.with good stirring. After stirring for 2 h, the solids were collected ona Buchner funnel, washed with water (3×250 mL) and dried under reducedpressure. The crude product isolated from several batches (135 g) wascombined and triturated with dichloromethane (4 L) for 6 h. Theresulting solid was collected on a Buchner funnel and dried underreduced pressure to give the title compound (121 g).

(e) 5-Acetyl-8-benzyloxy-1H-quinolin-2-one

To the product from step (b) (37.7 g, 186 mmol) was addedN,N-dimethylformamide (200 mL) and potassium carbonate (34.5 g, 250mmol) followed by benzyl bromide (31.8 g, 186 mmol). The mixture wasstirred at room temperature for 2.25 hour and then poured into saturatedsodium chloride (3.5 L) at 0° C. and stirred for 1 hour. The product wascollected and dried on a Buchner funnel for 1 hour, and the resultingsolids were dissolved in dichloromethane (2 L) and this mixture wasdried over sodium sulfate. The solution was filtered through a pad ofCelite which was then washed with dichloromethane (5×200 mL). Thecombined filtrate was then concentrated to dryness and the resultingsolids were triturated with ether (500 mL) for 2 h. The product wascollected on a Buchner funnel, washed with ether (2×250 mL) and driedunder reduced pressure to give the title compound (44 g) as a powder.

(d) 8-Benzyloxy-5-(2-bromoacetyl)-1H-quinolin-2-one

The product from step (c) (20.0 g, 68.2 mmol) was dissolved indichloromethane (200 mL) and cooled to 0° C. Boron trifluoride diethyletherate (10.4 mL, 82.0 mmol) was added via syringe and the mixture waswarmed to room temperature to give a thick suspension. The suspensionwas heated at 45° C. (oil bath) and a solution of bromine (11.5 g, 72.0mmol) in dichloromethane (100 mL) was added over 40 min. The mixture waskept at 45° C. for an additional 15 min and then cooled to roomtemperature. The mixture was concentrated under reduced pressure andthen triturated with 10% aqueous sodium carbonate (200 mL) for 1 hour.The solids were collected on a Buchner funnel, washed with water (4×100mL) and dried under reduced pressure. The product of two runs wascombined for purification. The crude product (52 g) was triturated with50% methanol in chloroform (500 mL) for 1 hour. The product wascollected on a Buchner funnel and washed with 50% methanol in chloroform(2×50 mL) and methanol (2×50 mL). The solid was dried under reducedpressure to give the title compound (34.1 g) as a powder.

Preparation 88-Benzyloxy-5-[(R)-2-bromo-1-(tert-butyldimethylsilanyloxy)ethyl]-1H-quinolin-2-one(a) 8-Benzyloxy-5-((R)-2-bromo-1-hydroxyethyl)-1H-quinolin-2-one

(R)-(+)-α,α-Diphenylprolinol (30.0 g, 117 mmol) and trimethylboroxine(11.1 mL, 78 mmol) were combined in toluene (300 mL) and stirred at roomtemperature for 30 min. The mixture was placed in a 150° C. oil bath andliquid was distilled off. Toluene was added in 20 mL aliquots anddistillation was continued for 4 h. A total of 300 mL toluene was added.The mixture was then cooled to room temperature. A 500 μL aliquot wasevaporated to dryness and weighed (246 mg) to determine that theconcentration of catalyst was 1.8 M.

8-Benzyloxy 5-(2-bromoacetyl)-1H-quinolin-2-one (90.0 g, 243 mmol) wasplaced under nitrogen and tetrahydrofuran (900 mL) was added followed bythe catalyst described above (1.8 M in toluene, 15 mL, 27 mmol). Thesuspension was cooled to −10±5° C. in an ice/isopropanol bath. Borane(1.0 M in THF, 294 mL, 294 mmol) was added over 4 h. The reaction wasthen stirred an additional 45 min at −10° C. and then methanol (250 mL)was added slowly. The mixture was concentrated under vacuum and theresidue was dissolved in boiling acetonitrile (1.3 L), filtered whilehot and then cooled to room temperature. The crystals were filtered,washed with acetonitrile and dried under vacuum to give the titlecompound (72.5 g, 196 mmol, 81% yield, 95% ee, 95% pure by HPLC).

(b)8-Benzyloxy-5-[(R)-2-bromo-1-(tert-butyldimethylsilanyloxy)ethyl]-1H-quinolin-2-one

To the product of step (b) (70.2 g, 189 mmol) was addedN,N-dimethylformamide (260 mL) and this mixture was cooled in an icebath under nitrogen, 2,6-Lutidine (40.3 g, 376 mmol) was added over 5min and then tert-butyldimethylsilyl trifluoromethanesulfonate (99.8 g,378 mmol) was added slowly while maintaining the temperature below 20°C. The mixture was allowed to warm to room temperature for 45 min.Methanol (45 mL) was added to the mixture dropwise over 10 min and themixture was partitioned between ethyl acetate/cyclohexane(1:1, 500 mL)and water/brine (1:1, 500 mL). The organics were washed twice more withwater/brine (1:1, 500 mL each). The combined organics were evaporatedunder reduced pressure to give a light yellow oil. Two separate portionsof cyclohexane (400 mL) were added to the oil and distillation continueduntil a thick white slurry was formed. Cyclohexane (300 mL) was added tothe slurry and the resulting white crystals were filtered, washed withcyclohexane (300 mL) and dried under reduced pressure to give the titlecompound (75.4 g, 151 mmol, 80% yield, 98.6% ee).

Preparation 9A8-Benzyloxy-5-[(R)-2-(N-benzylamino)-1-(tert-butyldimethylsilanyloxy)ethyl]-1H-quinolin-2-one

A stirred solution of the product of Preparation 8 (1.00 g, 2.05 mmol)and benzylamine (493 μL, 4.51 mmol) in DMSO (1.7 mL) was heated at 105°C. for 4 h. The reaction mixture was allowed to cool and was thendiluted with EtOAc (10 mL) and the organic layer was washed withsaturated aqueous ammonium chloride solution (5 mL) and 1N sodiumhydroxide (5 mL), dried (MgSO₄) and solvent removed under reducedpressure. The crude residue was purified by column chromatography (50%EtOAc/hexanes) to give the title compound (700 mg, 67%). MS m/z: [M+H⁺]calcd for C₃₁H₃₈N₂O₃Si 515.27; found 515.5.

Preparation 9B8-Benzyloxy-5-[(R)-2-(N-benzylamino)-1-(tert-butyldimethylsilanyloxy)ethyl]-1H-quinolin-2-one

To a 500 mL three-necked round-bottom flask was added8-benzyloxy-5-[(R)-2-bromo-1-(tert-butyldimethylsilanyloxy)ethyl]-1H-quinolin-2-one(43 g, 0.124 mol, about 95% chiral purity), 1-methyl-2-pyrrolidinone(210 mL) and benzylamine (28.3 g, 0.37 mol). The resulting mixture wasflushed with nitrogen and then stirred at 90° C. for 6 hours. Themixture was then cooled to room temperature and water (300 mL) and ethylacetate (300 mL) were added. The layers were separated and the organiclayer was washed with water (200 mL), a 1:1 mixture of water and aqueoussaturated sodium chloride solution (200 mL), and water (200 mL). Theorganic layer was then dried over magnesium sulfate, filtered andconcentrated under reduced pressure to provide the title compound as anorange oil.

To the orange oil was added heptane (200 mL) and ethyl acetate (200 mL)and the resulting mixture was heated to 65° C. to produce a clearsolution. This solution was cooled to room temperature and allowed tostand overnight (about 16 hours) at which time a precipitate had formed.The precipitate was collected by filtration to givestereochemically-impure title compound (8.85 g, 79.6% ee). The filtratewas concentrated under reduced pressure to give the title compound (38.6g, 99.4% ee). This material was combined with a previous batch ofmaterial (19.2 g, 99.5% ee) and heptane (250 mL) and ethyl acetate (100mL) were added. This mixture was heated to 80° C. (hazy to clearsolution) and then cooled to room temperature and allowed to standovernight. The resulting precipitate was collected by filtration toafford the title compound as a white solid (36.8 g. 98.4% ee, 99.9%chemical purity). The filtrate was concentrated under reduced pressureand the residue was dissolved in heptane (100 mL). The resulting solidswere collected to give the title compound as a tan solid (24 g, 100%chiral purity, 95% chemical purity).

Preparation 10A5-[(R)-2-Amino-1-(tert-butyldimethylsilanyloxy)ethyl]-8-hydroxy-1H-quinolin-2-one

A stirred solution of the product of Preparation 9A (3.16 g, 6.15 mmol)and palladium (10 wt. % (dry basis) on activated carbon) (1.58 g) inethanol (62 mL) was placed under an atmosphere of hydrogen for 24 h. Thereaction mixture was filtered through Celite, washed with methanol (15mL), and then the solvent was removed under reduced pressure to give thetitle compound as a solid (1.52 g, 4.55 mmol, 74%).

Preparation 10B5-[(R)-2-Amino-1-(tert-butyldimethylsilanyloxy)ethyl]-8-hydroxy-1H-quinolin-2-oneAcetic Acid Salt

8-Benzyloxy-5-[(R)-2-(N-benzylamino)-1-(tert-butyldimethylsilanyloxy)ethyl]-1H-quinolin-2-one(100 g, 194 mmol) and acetic acid (17.5 mL, 291 mmol) were dissolved inmethanol (1 L). The clear solution was purged with nitrogen and thenpalladium hydroxide on carbon (20 g, 20 wt. % Pd (dry basis), wet (about50% water)) was added. Hydrogen gas was bubbled through the stirredsolution at room temperature for 6 hours during which time a thickslurry developed. The reaction mixture was then purged with nitrogen andmethanol (1 L) was added. The resulting mixture was stirred for about 30minutes (to dissolve the product) and then the mixture was filteredthrough a pad of Celite. The filterate was concentrated under reducedpressure to a volume of about 500 mL and, to the resulting slurry, wasadded ethanol (500 mL). The resulting mixture was again concentratedunder reduced pressure to a volume of about 500 mL and the resultingprecipitate was collected by filtration and dried to provide the titlecompound as a yellow-white solid (65 g, 85% yield, >98% purity).

Preparation 11 Biphenyl-2-ylcarbamic Acid1-[2-(4-{[(R)-2-(tert-Butyldimethylsilanyloxy)-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-2-chloro-5-methoxy-phenylcarbamoyl)ethyl]piperidin-4-ylEster

To the product from Preparation 6 in a mixture of dichloromethane (0.5mL) and methanol (0.5 mL) was added the product of Preparation 10A(124.1 mg, 3.1 mmol) and the resulting mixture was stirred at roomtemperature for 1.5 h. Sodium triacetoxyborohydride (190.7 mg, 0.9 mmol)was added and the resulting mixture was stirred at room temperature for15 h. The reaction was quenched by adding water (about 0.2 mL) and themixture was concentrated in vacuo to give the title compound, which wasused without further purification. MS m/z 854.5 (M+H, expected 853.36for C₄₆H₅₆ClN₅O₇Si).

Preparation 12 Biphenyl-2-ylcarbamic Acid1-[2-(2-Chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)-ethyl]piperidin-4-ylEster

To a suspension of the product of Preparation 11 in dichloromethane (1.0mL, 0.3 M) added triethylamine trihydrofluoride (245 μL, 1.5 mmol). Thismixture was stirred at room temperature for 45 h and then the mixturewas concentrated in vacuo. The residue was dissolved in a mixture of DMF(0.5 mL), acetonitrile/water (1:1, with 0.1% TFA, 0.6 mL), TFA (0.3 mL)and acetonitrile (about 1 mL) and this mixture was purified byprep-RP-HPLC (gradient: 2 to 50% acetonitrile in water with 0.05% TFA).The appropriate fractions were collected and combined and lyophilized togive the ditrifluoroacetate salt of the title compound (100 mg, 34%yield, 98.7% pure by HPLC) as an off-whin. solid. MS m/z 740.5 (M+H,expected 739.28 for C₄₀H₄₂ClN₅O₇).

Example 1 Biphenyl-2-ylcarbamic Acid1-[2-(2-Chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)-ethyl]piperidin-4-ylEster 1,2-Ethanedisulfonic Acid Salt

A solution of 1,2-ethanedisulfonic acid dihydrate (3.8 mg, 0.02 mmol) inethanol 0.2 mL) was slowly added to a solution of the product ofPreparation 12 (1.4.3 mg, 0.02 mmol) in a 64:1 v/v mixture ofisopropanol and dichloromethane (1 mL). The resulting solution washeated at 45° C. to 50° C. for about 30 minutes. The mixture was thenslowly cooled to room temperature at which time the solution becameslightly cloudy. The solution was allowed to stand at ambienttemperature under a gentle stream of nitrogen overnight. The resultingprecipitate was collected by filtration and dried to provide the titlecompound as a white crystalline solid (13 mg, 72% yield).

Example 2 Biphenyl-2-ylcarbamic Acid1-[2-(2-Chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)-ethyl]piperidin-4-ylEster 1,2-Ethanedisulfonic Acid Salt

A solution of biphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(r)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)-ethyl]piperidin-4-ylester, (26.8mg, 0.0362 mmol) was prepared in ethanol (5.36 mL) andstirred at room temperature until complete dissolution was obtained (5min). A solution of 1,2-ethanedisulfonic acid dihydrate (8.2 mg, 0.0362mmol) in ethanol (0.2 mL) was slowly added to the first solution overapproximately one minute. The resulting suspension was stirred for fiveminutes then isolated by filtration under nitrogen. The resultingprecipitate was dried to provide the title compound as a white solid(28.5 mg, 85% yield).

Example 3 Biphenyl-2-ylcarbamic Acid1-[2-(2-Chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)-ethyl]piperidin-4-ylEster 1,2-Ethanedisulfonic Acid Salt

Biphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(r)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)-ethyl]piperidin-4-ylester (5 g, 6.75 mmol, >99% pure) was dissolved in isopropanol (100 mL),followed by addition of ethane disulfonic acid dihydrate (1.525 mg, 6.75mmol) dissolved in water (20 mL). The resulting slurry was stirred atroom temperature for 1 hour, and then at ˜30° C. overnight. The titlecompound (6.0 g) was isolated as a white powder. The product was heatedin 20% water in isopropanol (100 mL) at 30° C. for 48 hours. Aftercooling to room temperature, the resulting precipitate was isolated byfiltration and dried in air for 2 hours to yield the title compound (5.4g).

Preparation 13 Methyl 4-Acryloylamino-5-chloro-2-methoxybenzoate

To a 1-liter three-necked round-bottom flask equipped with overheadstirrer, temperature control and addition funnel was added methyl4-amino-5-chloro-2-methoxybenzoate (44.2 g, 200 mmol), dichloromethane(500 mL) and diisopropylethylamine (104.5 mL, 600 mmol). The resultingmixture was stirred at room temperature until the ingredients dissolvedand then the mixture was cooled to 0° C. Acryloyl chloride (16.25 mL,200 mmol) was then added dropwise while maintaining the internalreaction mixture temperature below 10° C. The total time for additionwas about 30 min. The reaction mixture was then slowly warmed from 0° C.to room temperature over a period of about 2 hours. Aqueous saturatedsodium bicarbonate solution (200 mL) and dichloromethane (200 mL) werethen added and this mixture was stirred for 15 min. and then the layerswere separated. The dichloromethane layer was washed with 1 Mhydrochloric acid (200 mL) and then concentrated under reduced pressureto about one-third its original volume resulting in a thick slurry. Theslurry was filtered and the filter cake was washed with dichloromethane(100 mL) and dried to provide the title compound as an off-white solid(36 g, 67% yield, >98% purity by HPLC).

Preparation 14 Methyl4-{3-[4-(Biphenyl-2-ylcarbamoyloxy)piperidin-1-yl]propionylamino}-5-chloro-2-methoxybenzoate

To a 1-liter three-necked round-bottom flask equipped with overheadstirrer, temperature control and reflux condensor was addedbiphenyl-2-ylcarbamic acid piperidin-4-yl ester (36.3 g, 122 mmol),dichloromethane (500 mL) and isopropanol (100 mL). The resulting mixturewas stirred at room temperature until the ingredients dissolved and thenthe product from Preparation 10 (30 g, 111.5 mmol) was added. Stirringwas continued at room temperature until the ingredients dissolved andthe mixture was then heated under reflux (50° C. to 55° C.) for 18hours. The reaction mixture was then cooled to room temperature andethanol (200 mL) was added. This mixture was concentrated under reducedpressure to a volume of about 150 mL resulting in a thick slurry. Theslurry was filtered and the filter cake was washed with ethanol (50 mL)and dried to provide the title compound as a white solid (58 g, 92%yield, 99.5% pure by HPLC).

Preparation 15 Biphenyl-2-ylcarbamic Acid1-[2-(2-Chloro-4-hydroxymethyl-5-methoxy-phenylcarbamoyl)ethyl]piperidin-4-ylEster

To a 2-liter round-bottom flask was added the product from Preparation14 (40 g, 70.8 mmol) and THF (400 mL). The resulting mixture was stirredat room temperature until the ingredients dissolved and then the flaskwas purged with nitrogen for 5 minutes. The mixture was then cooled 0°C. (internal temperature) and a 1 M solution of lithium aluminum hydridein THF (106 mL, 106 mmol) was added dropwise via an additional funnelwhile maintaining the internal reaction mixture temperature below 10° C.The total addition time was about 40 min. The reaction mixture was thenstirred for 1 hour at 0° C. and then 1 M sodium hydroxide (200 mL) wasadded while maintaining the internal reaction mixture temperature below15° C. The layers were then separated and the THF layer was washed withaqueous saturated sodium chloride solution (100 mL), dried over sodiumsulfate, filtered and concentrated under reduced pressure to provide thetitle compound as a white solid (38 g, 100% yield, 94% purity by HPLC).

Preparation 16 Biphenyl-2-ylcarbamic Acid1-[2-(2-Chloro-4-formyl-5-methoxyphenyl-carbamoyl)ethyl]piperidin-4-ylEster

To a 1-liter round-bottom flask was added the product from Preparation15 (28 g, 52 mmol) and dichloromethane (500 mL). The resulting mixturewas stirred at room temperature until the ingredients dissolved and thenactivated manganese(IV) oxide (45 g, 520 mmol) was added. The reactionmixture was stirred at room temperature under nitrogen for 12 hours andthen filtered through a pad of Celite. The mixture was then concentratedunder reduced pressure and the residue was dried overnight under vacuumto provide the title compound as a yellow solid (26 g, 93% yield, about93% purity by HPLC).

Preparation 17 Biphenyl-2-ylcarbamic Acid1-[2-(4-{[(R)-2-(tert-Butyldimethylsilanyloxy)-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-2-chloro-5-methoxy-phenylcarbamoyl)ethyl]piperidin-4-ylEster

To a 500 mL round-bottom flask was added the product from Preparation 16(6 g, 11.2 mmol) and dichloromethane (50 mL). The resulting mixture wasstirred at room temperature until the ingredients dissolved and then theproduct of Preparation 7 (6 g, 15.0 mmol) and dry methanol (50 mL) wereadded. This mixture was stirred at room temperature under nitrogen for 2hours (clear yellow to orange solution) and then the mixture was cooledto 0 to 5° C. Solid sodium triacetoxyborohydride (7.2 g, 34 mmol) wasadded in portions over a 10 minute period and then the reaction mixturewas slowly warmed from 0° C. to room temperature over a period of about2 hours. The mixture was then cooled to 0° C. and 1 M aqueous sodiumhydroxide solution (50 mL) and dichloromethane 150 mL) were added. Themixture was stirred thoroughly and then the layers were separated. Theorganic layer was washed with aqueous saturated sodium chloride solution(50 mL), filtered, dried over sodium sulfite, filtered and concentratedunder reduced pressure to give the title compound as a yellow solid(10.1 g, 100% yield, 87% purity by HPLC).

Example 4 Biphenyl-2-ylcarbamic Acid1-[2-(2-Chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)-ethyl]piperidin-4-ylEster 1,2-Ethanedisulfonic Acid Salt

To a 50 mL round-bottom flask was added the product from Preparation 17(2.0 g, 2.5 mmol) and dichloromethane (10 mL). The resulting mixture wasstirred at room temperature until the ingredients dissolved and thentriethylamine trihydrofluoride (1.2 mL, 7.5 mmol) was added and theresulting mixture was stirred at 25° C. for 20 hours. A solution of1,2-ethanedisulfonic acid dihydrate (0.56 g, 2.5 mmol) in methanol (10mL) was then added and this mixture was stirred at 30° C. for 2 hours atwhich time a thick white slurry had formed. The slurry was filteredslowly and the filter cake was washed with methanol (10 mL), air driedfor 2 hours and then dried overnight under vacuum to provide the titlecompound as a fine white powder (1.5 g, >98% purity by HPLC).

Example 5 Purification of Biphenyl-2-ylcarbamic Acid1-[2-(2-Chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)-ethyl]piperidin-4-ylEster 1,2-Ethanedisulfonic Acid Salt

To biphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)-ethyl]piperidin-4-ylester 1,2-ethanedisulfonic acid salt, prepared as in Example 4 (80 g)was added a solution of 20% water in isopropanol by volume (800 mL). Theresulting slurry was left at room temperature overnight and thenfiltered to provide the title compound having improved crystallinity andpurity (74 g).

Preparation 18 Seed Crystals of Biphenyl-2-ylcarbamic Acid1-[2-(2-Chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)-ethyl]piperidin-4-ylEster 1,2-Ethanedisulfonic Acid Salt

Step (a)

Biphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)-ethyl]piperidin-4-ylester 1,2-ethanedisulfonic acid salt, prepared as in Example 4 (100 mg)was dissolved in 13% water in methanol (20 mL) at ˜60° C. The resultingclear solution was allowed to cool to room temperature in a closedcontainer. After 48 hours, the resulting plate-like crystals wereisolated by filtration.

Step (b)

Biphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)-ethyl]piperidin-4-ylester 1,2-ethanedisulfonic acid salt, prepared as in Example 4 (1.0 g)was dissolved in 15% water in methanol (100 mL) at 60-65° C. The clear,stirred solution was allowed to cool to 30° C., and then the crystallineproduct of Step (a) (4.2 mg) was added. The solution was cooled to 20°C. and stirred for 2 hours. The resulting precipitate was isolated byfiltration and dried in air for 1 hour to provide the title compound(680 mg).

Step (c)

The procedure of Step (b) was repeated substituting the product of Step(b) (20 mg) for the product of step (a), to provide the title compound(690 mg).

Step(d)

Biphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)-ethyl]piperidin-4-ylester 1,2-ethanedisulfonic acid salt, prepared as in Example 4 (10 g)was dissolved in 15% water in methanol (1 L) at 60-65° C. The clear,stirred solution was allowed to cool to 30° C., and then the crystallineproduct of step (c) (4.2 mg) was added. The solution was cooled to 20°C. and stirred for 18 hours. The resulting precipitate was isolated byfiltration and dried in air for 2 hours to provide the title compound(5.5 g).

Example 6 Recrystallization of Biphenyl-2-ylcarbamic Acid1-[2-(2-Chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)-ethyl]piperidin-4-ylEster 1,2-Ethanedisulfonic Acid Salt Using Seed Crystals

To a 12 L round-bottom flask was added the product from Example 5 (60 g,64.5 mmol), water (0.9 L) and methanol (5.1 L). The resulting mixturewas heated from 25° C. to 61-65° C. with stirring until the ingredientsdissolved and stirred for an additional 20 minutes at 60-65° C. Themixture was allowed to cool to 30° C. and then the product ofPreparation 18 (2 g, 2.15 mmol) was added. This mixture was slowlycooled to 20° C. and the resulting slurry was stirred for an additional2 hours at 30° C. The product was filtered with methanol (500 mL) anddried in air for 2 hours and then in vacuo at 25-30° C. for 18 hours toprovide the title compound (43 g, 72% yield, 99.2% purity).

Example 7 Thermal Analysis

Differential scanning calorimetry (DSC) was performed using a TAInstruments Model Q-10 module with a Thermal Analyst controller. Datawere collected and analyzed using TA Instruments Thermal Solutionssoftware. A sample of about 1 mg was accurately weighed into an aluminumpan with lid. The sample was evaluated using a linear heating ramp of 5°C./min from ambient temperature to approximately 300° C. The DSC cellwas purged with dry nitrogen during use. A representative DSC trace fora sample of a crystalline 1,2-ethanedisulfonic acid salt ofbiphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester of Example 6 is shown in FIG. 1 and a representative DSC trace fora sample of Example 2 is shown in FIG. 2.

Thermogravimetric analysis (TGA) was performed using a TA InstrumentsModel Q-50 module equipped with high resolution capability. Data werecollected and analyzed using TA Instruments Thermal Solutions software.A sample weighing about 10 mg was placed onto a platinum pan and scannedwith a high resolution-heating rate from ambient temperature to 300° C.The balance and furnace chambers were purged with nitrogen flows duringuse. A representative TGA trace for a sample of a crystalline1,2-ethanedisulfonic acid salt of biphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester of Example 6 is shown in FIG. 1.

The DSC traces demonstrate that a 1,2-ethanedisulfonic acid salt of thepresent invention has excellent thermal stability with melting points atabout 239° C. and at about 219° C., respectively, and no thermaldecomposition below about 200° C.

Example 8 Powder X-Ray Diffraction

Powder x-ray diffraction patterns were obtained with a Thermo ARL X-RayDiffractometer Model X'TRA (Thermo ARL SA, Switzerland) using Cu Kαradiation at 1.542 Å (45 kV, 40 mA) with a Si(Li) solid-state detector.The analysis was typically performed at a scan rate of 2°/min with astep size of 0.03° per point over a range of 2 to 30° in two-thetaangle. Samples, either as received or ground to a fine powder, weregently packed into a custom small-volume insert designed to fit into theinstrument top-loading sample cup for analysis. The instrument wascalibrated weekly to a silicon metal standard, within ±0.02° two-thetaangle. A representative PXRD patterns for a sample of a crystalline1,2-ethanedisulfonic acid salt of biphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester of Example 6 is shown in FIG. 3 and the pattern for a sample ofExample 2 is shown in FIG. 4.

Example 9 Infrared Analysis

The infrared (IR) absorption spectrum was determined over the frequencyrange 4000 to 675 cm⁻¹ using an Avatar 360 FT-IR spectrometer equippedwith a Nicolet attenuated total reflection (ATR) sample holder. Arepresentative IR absorption spectrum for a sample of a crystalline1,2-ethanedisulfonic acid salt of biphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester for a sample of Example 6 had significant absorption bands at704±1, 748±1, 768±1, 841±1, 900±1, 1055±1, 1104±1, 1166±1, 1218±1,1294±1, 1408±1, 1522±1, 1609±1, 1655±1, and 1701±1, as illustrated inFIG. 5.

Example 10 Dynamic Moisture Sorption Assessment

A dynamic moisture sorption (DMS) assessment (also known as a moisturesorption-desorption profile) was performed for a hand ground sample of acrystalline 1,2-ethanedisulfonic acid salt of biphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester of Preparation 18 using VTI atmospheric microbalance, SGA-100system (VTI Corp., Hialeah, Fla. 33016), A sample size of approximately10 mg was used and the humidity was set at the ambient value at thestart of the analysis. A typical DMS analysis consisted of three scans:ambient to 2% relative humidity (RH), 2% RH to 90% RH, 90% RH to 5% RHat a scan rate of 5% RH/step. The mass was measured every two minutesand the RH was changed to the next value (+/−5% RH) when the mass of thesample was stable to within 0.01% for 5 consecutive points. Arepresentative DMS trace is shown in FIG. 6.

The DMS trace demonstrates that a 1,2-ethanedisulfonic acid salt of thepresent invention has a reversible sorptionidesorption profile withmoderate (<9%) hygroscopicity, The salt has less than 2.5% weight gainin the humidity range of 40% RH to 75% RH. The reversible moisturesorption/desorption profile demonstrates that a crystalline salt of thepresent invention possesses an acceptable hygroscopicity and is notdeliquescent.

Example 11 Elemental Analysis and Counterion Ratio

The following elemental percentages of carbon, hydrogen, nitrogen, andsulfur of a sample of a crystalline 1,2-ethanedisulfonic acid salt ofbiphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester of Example 6 were determined by combustion analysis using a FlashEA 1112 Elemental Analyzer (GE Elantech, Lakewood, N.J.): carbon 52.95%,hydrogen 5.43%, nitrogen 6.83%, and sulfur 6.87%. The weight percentageof 1,2-ethanedisulfonic acid in the crystalline sample, calculated fromthe measured weight percentage of sulfur, is 20.4%, which is equal tothe theoretical weight percentage of 20.4%, providing a counterion ratioof 1:1.

Preparation A Cell Culture and Membrane Preparation From CellsExpressing Human β₁, β₂ or β₃ Adrenergic Receptors

Chinese hamster ovarian (CHO) cell lines stably expressing cloned humanβ₁, β₂ or β₃ adrenergic receptors, respectively, were grown to nearconfluency in Hams F-12 media with 10% FBS in the presence of 500 μg/mLGeneticin. The cell monolayer was lifted with 2 mM EDTA in PBS. Cellswere pelleted by centrifugation at 1,000 rpm, and cell pellets wereeither stored frozen at −80° C. or membranes were prepared immediatelyfor use. For preparation of β₁ and β₂ receptor expressing membranes,cell pellets were re-suspended in lysis buffer (10 mM HEPES/HCl, 10 mMEDTA, pH 7.4 at 4° C.) and homogenized using a tight-fitting Dounceglass homogenizer (30 strokes) on ice. For the more protease-sensitiveβ₃ receptor expressing membranes, cell pellets were homogenated lysisbuffer (10 mM Tris/HCl, pH 7.4) supplemented with one tablet of“Complete Protease Inhibitor Cocktail Tablets with 2 mM EDTA” per 50 mLbuffer (Roche Catalog No. 1697498, Roche Molecular Biochemicals,Indianapolis, Ind.). The homogenate was centrifuged at 20,000×g, and theresulting pellet was washed once with lysis buffer by re-suspension andcentrifugation as above. The final pellet was then re-suspended inice-cold binding assay buffer (75 mM Tris/HCl pH 7.4, 12.5 mM MgCl₂, 1mM EDTA). The protein concentration of the membrane suspension wasdetermined by the methods described in Lowry et al., 1951, Journal ofBiological Chemistry, 193, 265; and Bradford, Analytical Biochemistry,1976, 72, 248-54. All membranes were stored frozen in aliquots at −80°C. or used immediately.

Preparation B Cell Culture and Membrane Preparation from CellsExpressing Human M₁, M₂, M₃ and M₄ Muscarinic Receptors

CHO cell lines stably expressing cloned human hM₁, hM₂, hM₃ and hM₄muscarinic receptor subtypes, respectively, were grown to nearconfluency in HAM's F-12 media supplemented with 10% FBS and 250 μg/mLGeneticin. The cells were grown in a 5% CO₂, 37° C. incubator and liftedwith 2 mM EDTA in dPBS. Cells were collected by 5 minute centrifugationat 650×g, and cell pellets were either stored frozen at −80° C. ormembranes were prepared immediately for use. For membrane preparation,cell pellets were resuspended in lysis buffer and homogenized with aPolytron PT-2100 tissue disrupter (Kinematica AG; 20 seconds×2 bursts),Crude membranes were centrifuged at 40,000×g for 15 minutes at 4° C. Themembrane pellet was then resuspended with re-suspension buffer andhomogenized again with the Polytron tissue disrupter. The proteinconcentration of the membrane suspension was determined by the methoddescribed in Lowry et al., 1951, Journal of Biochemistry, 193, 265. Allmembranes were stored frozen in aliquots at −80° C. or used immediately.Aliquots of prepared hM₅ receptor membranes were purchased directly fromPerkin Elmer and stored at −80° C. until use.

Assay Test Procedure A Radioligand Binding Assay for Human β₁, β₂ and β₃Adrenergic Receptors

Binding assays were performed in 96-well microtiter plates in a totalassay volume of 100 μL with 10-15 μg of membrane protein containing thehuman β₁, β₂ or β₃ adrenergic receptors in assay buffer (75 mM Tris/HClpH 7.4 at 25° C., 12.5 mM MgCl₂, 1 mM EDTA, 0.2% BSA). Saturationbinding studies for determination of K_(d) values of the radioligandwere done using [³H]-dihydroalprenolol (NET-720, 100 Ci/mmol,PerkinElmer Life Sciences Inc., Boston, Mass.) for the β₁ and β₂receptors and [¹²⁵I]-(−)-iodocyanopindolol (NEX-189, 220 Ci/mmol,PerkinElmer Life Sciences Inc., Boston, Mass.) at 10 or 11 differentconcentrations ranging from 0.01 nM to 20 nM. Displacement assays fordetermination of K_(i) values of test compounds were done with[³H]-dihydroalprenolol at 1 nM and [¹²⁵I]-(−)-iodocyanopindolol at 0.5nM for 10 or 11 different concentrations of test compound ranging from10 pM to 10 μM. Non-specific binding was determined in the presence of10 μM propranolol. Assays were incubated for 1 hour at 37° C., and thenbinding reactions were terminated by rapid filtration over GF/B for theβ₁ and β₂ receptors or GF/C glass fiber filter plates for the β3receptors (Packard BioScience Co., Meriden, Conn.) presoaked in 0.3%polyethyleneimine. Filter plates were washed three times with filtrationbuffer (75 mM Tris/HCl pH 7.4 at 4° C., 12.5 mM MgCl₂, 1 mM EDTA) toremove unbound radioactivity. The plates were then dried and 50 μL ofMicroscint-20 liquid scintillation fluid (Packard BioScience Co.,Meriden, Conn.) was added and plates were counted in a Packard Topcountliquid scintillation counter (Packard BioScience Co., Meriden, Conn.),Binding data were analyzed by nonlinear regression analysis with theGraphPad Prism Software package (GraphPad Software, Inc., San Diego,Calif.) using the 3-parameter model for one-site competition. The curveminimum was fixed to the value for nonspecific binding, as determined inthe presence of 10 μM propranolol. K_(i) values for test compounds werecalculated from observed IC₅₀ values and the K_(d) value of theradioligand using the Cheng-Prusoff equation (Cheng Y, and Prusoff W H.,Biochemical Pharmacology, 1973, 22, 23, 3099-108).

In this assay, a lower K_(i) value indicates that a test compound has ahigher binding affinity for the receptor tested, When tested in thisassay, the compound of formula I was found to have a K_(i) value of lessthan 10 nM for the human β₂ adrenergic receptor.

Assay Test Procedure B Radioligand Binding Assay for MuscarinicReceptors

Radioligand binding assays for cloned human muscarinic receptors wereperformed in 96-well microtiter plates in a total assay volume of 100μL. CHO cell membranes stably expressing either the hM₁, hM₂, hM₃, hM4or hM₅ muscarinic subtype were diluted in assay buffer to the followingspecific target protein concentrations (μg/well): 10 μg for hM₁, 10-15μg for hM₂, 10-20 μg for hM₃, 10-20 μg for hM₄, and 10-12 μg for hM₅ toget similar signals (cpm). The membranes were briefly homogenized usinga Polytron tissue disruptor (10 seconds) prior to assay plate addition.Saturation binding studies for determining K_(D) values of theradioligand were performed using L-[N-methyl-³H]scopolamine methylchloride ([³H]-NMS) (TRK666, 84.0 Ci/mmol, Amersham Pharmacia Biotech,Buckinghamshire, England) at concentrations ranging from 0.001 nM to 20nM. Displacement assays for determination of K_(i) values of testcompounds were performed with [³H]-NMS at 1 nM and eleven different testcompound concentrations. The test compounds were initially dissolved toa concentration of 400 μM in dilution buffer and then serially diluted5× with dilution buffer to final concentrations ranging from 10 pM to100 μM. The addition order and volumes to the assay plates were asfollows: 25 μL radioligand, 25 μL, diluted test compound, and 50 μLmembranes. Assay plates were incubated for 60 minutes at 37° C. Bindingreactions were terminated by rapid filtration over GF/B glass fiberfilter plates (PerkinElmer Inc., Wellesley, Mass.) pre-treated in 1%BSA. Filter plates were rinsed three times with wash buffer (10 mMHEPES) to remove unbound radioactivity. The plates were then air driedand 50 μL Microscint-20 liquid scintillation fluid (PerkinElmer Inc.,Wellesley, Mass.) was added to each well. The plates were then countedin a PerkinElmer Topcount liquid scintillation counter (PerkinElmerInc., Wellesley, Mass.). Binding data were analyzed by nonlinearregression analysis with the GraphPad Prism Software package (GraphPadSoftware, Inc., San Diego, Calif.) using the one-site competition model.K_(i) values for test compounds were calculated from observed IC₅₀values and the K_(D) value of the radioligand using the Cheng-Prusoffequation (Cheng Y; Prusoff W H. (1973) Biochemical Pharmacology,22(23):3099-108). K_(i) values were converted to pK_(i) values todetermine the geometric mean and 95% confidence intervals. These summarystatistics were then converted back to K_(i) values for data reporting.

In this assay, a lower K_(i) value indicates that the test compound hasa higher binding affinity for the receptor tested. When tested in thisassay, the compound of formula I was found to have a K_(i) value of lessthan 10 nM for the human M₂ and M₃ muscarinic receptors.

Assay Test Procedure C Whole-Cell cAMP Flashplate Assay in CHO CellLines Heterologously Expressing Human β₁, β₂ or β₃ Adrenergic Receptors

cAMP assays were performed in a radioimmunoassay format using theFlashplate Adenylyl Cyclase Activation Assay System with [¹²⁵I]-cAMP(NEN SMP004, PerkinElmer Life Sciences Inc., Boston, Mass.), accordingto the manufacturers instructions. For the determination of β receptoragonist potency (EC₅₀), CHO-K1 cell lines stably expressing cloned humanβ₁, β₂ or β₃ adrenergic receptors were grown to near confluency in HAM'sF-12 media supplemented with 10% FBS and Geneticin (250 μg/mL). Cellswere rinsed with PBS and detached in dPBS (Dulbecco's Phosphate BufferedSaline, without CaCl₂ and MgCl₂) containing 2 mM EDTA or Trypsin-EDTAsolution (0.05% trypsin/0.53 mM EDTA). After counting cells in Coultercell counter, cells were pelleted by centrifugation at 1,000 rpm andre-suspended in stimulation buffer containing IBMX (PerkinElmer Kit)pre-warmed to room temperature to a concentration of 1.6×10⁶ to 2.8×10⁶cells/mL. About 60,000 to 80,000 cells per well were used in this assay.Test compounds (10 mM DMSO) were diluted into PBS containing 0.1% BSA inBeckman Biomek-2000 and tested at 11 different concentrations rangingfrom 100 μM to 1 pM. Reactions were incubated for 10 min at 37° C. andstopped by adding 100 μL of cold detection buffer containing [¹²⁵I]-cAMP(NEN SMP004, PerkinElmer Life Sciences, Boston, Mass.). The amount ofcAMP produced (pmol/well) was calculated based on the counts observedfor the samples and cAMP standards as described in the manufacturer'suser manual. Data were analyzed by nonlinear regression analysis withthe GraphPad Prism Software package (GraphPad Software, Inc., San Diego,Calif.) with the sigmoidal equation. The Cheng-Prusoff equation (ChengY, and Prusoff W H., Biochemical Pharmacology, 1973, 22, 23, 3099-108)was used to calculate the EC50 values.

In this assay, a lower EC₅₀ value indicates that the test compound has ahigher functional activity at the receptor tested. When tested in thisassay, the compound of formula I was found to have an EC₅₀ value of lessthan 10 nM for the human β₂ adrenergic receptor.

Assay Test Procedure D Functional Assays of Antagonism for MuscarinicReceptor Subtypes A. Blockade of Agonist-Mediated [³⁵S]GTPγS Binding

The functional potency of a test compound was determined by measuringthe ability of the compound to block oxotremorine-stimulated [³⁵S]GTPγSbinding in CHO-K1 cells expressing the hM₂ receptor.

At the time of use, frozen membranes were thawed and then diluted inassay buffer with a final target tissue concentration of 5-10 μg proteinper well. The membranes were briefly homogenized using a PolytronPT-2100 tissue disrupter and then added to the assay plates.

The EC₉₀ value (effective concentration for 90% maximal response) forstimulation of [³⁵S]GTPγS binding by the agonist oxotremorine wasdetermined in each experiment.

To determine the ability of a test compound to inhibitoxotremorine-stimulated [³⁵S]GTPγS binding, the following was added toeach well of 96 well plates: 25 μL of assay buffer with [³⁵S]GTPγS (0.4nM), 25 μL of oxotremorine(EC₉₀) and GDP (3 uM), 25 μL of diluted testcompound and 25 μL CHO cell membranes expressing the hM₂ receptor. Theassay plates were then incubated at 37° C. for 60 minutes. The assayplates were filtered over 1% BSA-pretreated GF/B filters using aPerkinEimer 96-well harvester. The plates were rinsed with ice-cold washbuffer for 3×3 seconds and then air or vacuum dried. Microscint-20scintillation liquid (50 μL) was added to each well, and each plate wasseated and radioactivity counted on a Topcounter (PerkinElmer). Datawere analyzed by nonlinear regression analysis with the GraphPad PrismSoftware package (GraphPad Software, Inc., San Diego, Calif.) using thenon-linear regression, one-site competition equation. The Cheng-Prusoffequation was used to calculate the K_(i), using the IC₅₀ values of theconcentration-response curve for the test compound and the oxotremorineconcentration in the assay as the K_(D) and [L], ligand concentration,respectively.

In this assay, a lower K_(i) value indicates that the test compound hasa higher functional activity at the receptor tested. When tested in thisassay, the compound of formula I was found to have a K_(i) value of lessthan about 10 nM for blockade of oxotremorine-stimulated [^(S)S]GTPγSbinding in CHO-K1 cells expressing the hM₂ receptor.

B. Blockade of Agonist-Mediated Calcium Release via FLIPR Assays

Muscarinic receptor subtypes (M₁, M₃ and M₅ receptors), which couple toG_(q) proteins, activate the phospholipase C (PLC) pathway upon agonistbinding to the receptor. As a result, activated PLC hydrolyzesphosphatyl inositol diphosphate (PIP₂) to diacylglycerol (DAG) andphosphatidyl-1,4,5-triphosphate (IP₃), which in turn generates calciumrelease from intracellular stores, i.e., endoplasmic and sarcoplasmicreticulum. The FLIPR (Molecular Devices, Sunnyvale, Calif.) assaycapitalizes on this increase in intracellular calcium by using a calciumsensitive dye (Fluo-4AM, Molecular Probes, Eugene, Oreg.) thatfluoresces when free calcium binds. This fluorescence event is measuredin real time by the FLIPR, which detects the change in fluorescence froma monolayer of cells cloned with human M₁ and M₃, and chimpanzee M₅receptors. Antagonist potency can be determined by the ability ofantagonists to inhibit agonist-mediated increases in intracellularcalcium.

For FLIPR calcium stimulation assays, CHO cells stably expressing thehM₁, hM₃ and cM₅ receptors were seeded into 96-well FLIPR plates thenight before the assay was done. Seeded cells were washed twice byCellwash (MTX Labsystems, Inc.) with FLIPR buffer (10 mM HEPES, pH 7.4,2 mM calcium chloride, 2.5 mM probenecid in Hank's Buffered SaltSolution (HBSS) without calcium and magnesium) to remove growth mediaand leaving 50 μL/well of FLIPR buffer. The cells were then incubatedwith 50 μL/well of 4 μM FLUO-4AM (a 2× solution was made) for 40 minutesat 37° C., 5% carbon dioxide. Following the dye incubation period, cellswere washed two times with FLIPR buffer, leaving a final volume of 50μL/well.

To determine antagonist potency, the dose-dependent stimulation ofintracellular Ca²⁺ release for oxotremorine was first determined so thatantagonist potency can later be measured against oxotremorinestimulation at an EC₉₀ concentration. Cells were first incubated withcompound dilution buffer for 20 minutes, followed by agonist addition,which was performed by the FLIPR. An EC₉₀ value for oxotremorine wasgenerated according to the method detailed in the FLIPR measurement anddata reduction section below, in conjunction with the formulaEC_(F)=((F/100−F)̂1/H) .EC₅₀. An oxotremorine concentration of 3×EC_(F)was prepared in stimulation plates such that an EC₉₀ concentration ofoxotremorine was added to each well in the antagonist inhibition assayplates.

The parameters used for the FLIPR were: exposure length of 0.4 seconds,laser strength of 0.5 watts, excitation wavelength of 488 nm, andemission wavelength of 550 nm. Baseline was determined by measuring thechange in fluorescence for 10 seconds prior to addition of agonist.Following agonist stimulation, the FLIPR continuously measured thechange of fluorescence every 0.5 to 1 second for 1.5 minutes to capturethe maximum fluorescence change.

The change of fluorescence was expressed as maximum fluorescence minusbaseline fluorescence for each well. The raw data was analyzed againstthe logarithm of drug concentration by nonlinear regression withGraphPad Prism (GraphPad Software, Inc., San Diego, Calif.) using thebuilt-in model for sigmoidal dose-response. Antagonist K_(i) values weredetermined by Prism using the oxotremorine EC₅₀ value as the K_(D) andthe oxotremorine EC₉₀ for the ligand concentration according to theCheng-Prusoff equation (Cheng & Prusoff, 1973).

In this assay, a lower K_(i) value indicates that the test compound hasa higher functional activity at the receptor tested. When tested in thisassay, the compound of formula I was found to have a K_(i) value of lessthan about 10 nM for blockade of agonist-mediated calcium release in CHOcells stably expressing the hM₁, hM3 and cM₅ receptors.

Assay Test Procedure E Whole-Cell cAMP Flashplate Assay with a LungEpithelial Cell Line Endogenously Expressing Human β₂ AdrenergicReceptor

For the determination of agonist potencies and efficacies (intrinsicactivities) in a cell line expressing endogenous levels of the β₂adrenergic receptor, a human lung epithelial cell line (BEAS-2B) wasused (ATCC CRL-9609, American Type Culture Collection, Manassas, Va.)(January B, et al., British Journal of Pharmacology, 1998, 123, 4,701-11). Cells were grown to 75-90% confluency in complete, serum-freemedium (LHC-9 MEDIUM containing Epinephrine and Retinoic Acid, cat#181-500, Biosource International, Camarillo, Calif.). The day beforethe assay, medium was switched to LHC-8 (no epinephrine or retinoicacid, cat #141-500, Biosource International, Camarillo, Calif.).

cAMP assays were performed in a radioimmunoassay format using theFlashplate Adenylyl Cyclase Activation Assay System with [¹²⁵I]-cAMP(NEN SMP004, PerkinElmer Life Sciences Inc., Boston, Mass.), accordingto the manufacturers instructions.

On the day of the assay, cells were rinsed with PBS, lifted by scrapingwith 5 mM EDTA in PBS, and counted. Cells were pelleted bycentrifugation at 1,000 rpm and re-suspended in stimulation bufferpre-warmed to 37° C. at a final concentration of 600,000 cells/mL. Cellswere used at a final concentration of 100,000 to 120,000 cells/well inthis assay. Test compounds were serially diluted into assay butler (75mM Tris/HCl pH 7.4 at 25° C., 12.5 mM MgCl₂, 1 mM EDTA, 0.2% BSA) inBeckman Biomek-2000. Test compounds were tested in the assay at 11different concentrations, ranging from 10 μM to 10 pM. Reactions wereincubated for 10 min at 37° C. and stopped by addition of 100 μL ofice-cold detection buffer. Plates were sealed, incubated over night at4° C. and counted the next morning in a Topcount scintillation counter(Packard BioScience Co., Meriden, Conn.). The amount of cAMP producedper of reaction was calculated based on the counts observed for samplesand cAMP standards, as described in the manufacturer's user manual. Datawere analyzed by nonlinear regression analysis with the GraphPad PrismSoftware package (GraphPad Software, Inc., San Diego, Calif.) using the4-parameter model for sigmoidal dose-response.

In this assay, a lower EC₅₀ value indicates that the test compound has ahigher functional activity at the receptor tested. When tested in thisassay, the compound of formula I was found to have an EC₅₀ value of lessthan about 10 nM for the β₂ adrenergic receptor.

Assay Test Procedure F Duration Bronchoprotection in Guinea Models ofAcetylcholine-Induced or Histamine-Induced Bronchoconstriction

These in viva assays were used to assess the bronchoprotective effectsof test compounds exhibiting both muscarinic receptor antagonist and ∃₂adrenergic receptor agonist activity. To isolate muscarinic antagonistactivity in the acetylcholine-induced bronchoconstriction model, theanimals were administered propanolol, a compound that blocks β receptoractivity, prior to the administration of acetylcholine. Duration ofbronchoprotection in the histamine-induced bronchoconstriction modelreflects ∃₂ adrenergic receptor agonist activity.

Groups of 6 male guinea pigs (Duncan-Hartley (HsdPoc:DH) Harlan,Madison, Wis.) weighing between 250 and 350 g were individuallyidentified by cage cards. Throughout the study, animals were allowedaccess to food and water ad libitum.

Test compounds were administered via, inhalation over 10 minutes in awhole-body exposure dosing chamber (R&S Molds, San Carlos, Calif.). Thedosing chambers were arranged so that an aerosol was simultaneouslydelivered to 6 individual chambers from a central manifold. Guinea pigswere exposed to an aerosol of a test compound or vehicle (WFI). Theseaerosols were generated from aqueous solutions using an LC StarNebulizer Set (Model 22F51, PARI Respiratory Equipment, Inc, Midlothian,Va.) driven by a mixture of gases (CO₂=5%, O₂=21% and N₂=74%) at apressure of 22 psi. The gas flow through the nebutizer at this operatingpressure was approximately 3 L/minute. The generated aerosols weredriven into the chambers by positive pressure. No dilution air was usedduring the delivery of aerosolized solutions. During the 10 minutenebulization, approximately 1.8 mL of solution was nebulized. This valuewas measured gravimetrically by comparing pre-and post-nebulizationweights of the filled nebulizer.

The bronchoprotective effects of test compounds administered viainhalation were evaluated using whole body plethysmography at 1.5, 24,48 and 72 hours post-dose.

Forty-five minutes prior to the start of the pulmonary evaluation, eachguinea pig was anesthetized with an intramuscular injection of ketamine(43.75 mg/kg), xylazine (3.50 mg/kg) and acepromazine (1.05 mg/kg).After the surgical site was shaved and cleaned with 70% alcohol, a 2-3cm midline incision of the ventral aspect of the neck was made. Then,the jugular vein was isolated and cannulated with a saline-filledpolyethylene catheter (PE-50, Becton Dickinson, Sparks, Md.) to allowfor intravenous infusions of acetylcholine (Ach) or histamine in saline.The trachea was then dissected free and cannulated with a 14 G teflontube (#NE-014, Small Parts, Miami Lakes, Fla.). If required, anesthesiawas maintained by additional intramuscular injections of theaforementioned anesthetic mixture. The depth of anesthesia was monitoredand adjusted if the animal responds to pinching of its paw or if therespiration rate was greater than 100 breaths/minute.

Once the cannulations were completed, the animal was placed into aplethysmograph (#PLY3114, Buxco Electronics, Inc., Sharon, Conn.) and anesophageal pressure cannula (PE-160, Becton Dickinson, Sparks, Md.) wasinserted to measure pulmonary driving pressure (pressure). The teflontracheal tube was attached to the opening of the plethysmograph to allowthe guinea pig to breathe room air from outside the chamber. The chamberwas then sealed. A heating lamp was used to maintain body temperatureand the guinea pig's lungs were inflated 3 times with 4 mL of air usinga 10 mL calibration syringe (#5520 Series, Hans Rudolph, Kansas City,Mo.) to ensure that the lower airways did not collapse and that theanimal did not suffer from hyperventilation.

Once it was determined that baseline values were within the range of0.3-0.9 mL/cm H₂O for compliance and within the range of 0.1-0199 cmH₂O/mL per second for resistance, the pulmonary evaluation wasinitiated. A Buxco pulmonary measurement computer program enabled thecollection and derivation of pulmonary values.

Starting this program initiated the experimental protocol and datacollection. The changes in volume over time that occur within theplethysmograph with each breath were measured via a Buxco pressuretransducer. By integrating this signal over time, a measurement of flowwas calculated for each breath. This signal, together with the pulmonarydriving pressure changes, which were collected using a Sensym pressuretransducer (#TRD4100), was connected via a Buxco (MAX 2270) preamplifierto a data collection interface (#'s SFT3400 and SFT3813). All otherpulmonary parameters were derived from these two inputs.

Baseline values were collected for 5 minutes, after which time theguinea pigs were challenged with Ach or histamine. When evaluating themuscarinic antagonist effects, propanolol (5 mg/Kg, iv) (Sigma-Aldrich,St, Louis, Mo.) was administered 15 minutes prior to challenge with Ach.Ach (Sigma-Aldrich, Si. Louis, Mo.) (0.1 mg/mL) was infusedintravenously for 1 minute from a syringe pump (sp210iw, World PrecisionInstruments, Inc., Sarasota, Fla.) at the following doses and prescribedtimes from the start of the experiment: 1.9 μg/minute at 5 minutes, 3.8μg/minute at 10 minutes, 7.5 μg/minute at 15 minutes, 15.0 μg/minute at20 minutes, 30 μg/minute at 25 minutes and 60 μg/minute at 30 minutes.Alternatively, bronchoprotection of test compounds was assessed in theacetylcholine challenge model without pretreatment with a beta blockingcompound.

When evaluating the β₂ adrenergic receptor agonist effects of testcompounds, histamine (25 μg/mL) (Sigma-Aldrich, St. Louis, Mo.) wasinfused intravenously for 1 minute from a syringe pump at the followingdoses and prescribed times from the start of the experiment: 0.5μg/minute at 5 minutes, 0.9 μg/minute at 10 minutes, 1.9 μg/minute at 15minutes, 3.8 μg/minute at 20 minutes, 7.5 μg/minute at 25 minutes and 15μg/minute at 30 minutes. If resistance or compliance had not returned tobaseline values at 3 minutes following each Ach or histamine dose, theguinea pig's lungs were inflated 3 times with 4 mL of air from a 10 mLcalibration syringe. Recorded pulmonary parameters include respirationfrequency (breaths/minute), compliance (mL/cm H₂O) and pulmonaryresistance (cm H₂O/mL per second). Once the pulmonary functionmeasurements were completed at minute 35 of this protocol, the guineapig was removed from the plethysmograph and euthanized by carbon dioxideasphyxiation.

The data were evaluated in one of two ways:

(a) Pulmonary resistance (R_(L), cm H₂O/mL per second) was calculatedfrom the ratio of “change in pressure” to “the change in flow.” TheR_(L) response to ACh (60 μg/min, IH) was computed for the vehicle andthe test compound groups. The mean ACh response in vehicle-treatedanimals, at each pre-treatment time, was calculated and used to compute% inhibition of ACh response, at the corresponding pre-treatment time,at each test compound dose. Inhibition dose-response curves for ‘R_(L)’were fitted with a four parameter logistic equation using GraphPadPrism, version 3.00 for Windows (GraphPad Software, San Diego, Calif.)to estimate bronchoprotective ID₅₀ (dose required to inhibit the ACh (60μg/min) bronchocontrictor response by 50%). The equation used was asfollows:

Y=Min+(Max−Min)/(1+10^(((log ID50−X)*Hillslope)))

where X is the logarithm of dose, Y is the response (% Inhibition of AChinduced increase in R_(L)). Y starts at Min and approachesasymptotically to Max with a sigmoidal shape.

(b) The quantity PD₂, which is defined as the amount of Ach or histamineneeded to cause a doubling of the baseline pulmonary resistance, wascalculated using the pulmonary resistance values derived from the flowand the pressure over a range of Ach or histamine challenges using thefollowing equation (derived from the equation used to calculate PC₂₀values in the clinic (see Am. Thoracic Sac, 2000):

${PD}_{2} = {{antilog}\left\lbrack {{\log \; C_{1}} + \frac{\left( {{\log \; C_{2}} - {\log \; C_{1}}} \right)\left( {{2\; R_{0}} - R_{1}} \right)}{R_{2} - R_{1}}} \right\rbrack}$

where:

-   -   C₁=concentration of Ach or histamine preceding C₂    -   C₂=concentration of Ach or histamine resulting in at least a        2-fold increase in pulmonary resistance (R_(L))    -   R₀=Baseline R_(L) value    -   R₁=R_(L) value after C₁    -   R₂=R_(L) value after C₂

Statistical analysis of the data was performed using a twotailed—Students t-test. A P-value<0.05 was considered significant.

When tested in this assay, the compound of formula I produced adose-dependent bronchoprotective effect against MCh-inducedbronchoconstriction and His-induced bronchoconstriction. Additionally,the compound of formula I had a duration (PD T_(1/2)) ofbronchoprotective activity of at least about 24 hours in this assay.

While the present invention has been described with reference tospecific aspects or embodiments thereof, it will be understood by thoseof ordinary skilled in the art that various changes can be made orequivalents can be substituted without departing from the true spiritand scope of the invention. Additionally, to the extent permitted byapplicable patent statutes and regulations, all publications, patentsand patent applications cited herein are hereby incorporated byreference in their entirety to the same extent as if each document hadbeen individually incorporated by reference herein.

1-24. (canceled)
 25. A process for preparing a crystalline1,2-ethanedisulfonic acid salt of biphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester; the process comprising contacting biphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester with 1,2-ethanedisulfonic acid or a hydrate thereof.
 26. A processfor preparing a crystalline 1,2-ethanedisulfonic acid salt ofbiphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester, the process comprising: (a) contacting a compound of formula II:

wherein R^(1a), R^(1b) and R^(1c) are independently selected from C₁₋₄alkyl, phenyl, —C₁₋₄ alkyl-(phenyl), or one of R^(1a), R^(1b) and R^(1c)is —O—(C₁₋₄ alkyl); with fluoride ion; and (b) contacting the productfrom step (a) with 1,2-ethanedisulfonic acid or a hydrate thereof; toform a crystalline 1,2-ethanedisulfonic acid salt ofbiphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester, wherein step (a) and (b) are conducted in the same reactionvessel without isolation of the product of step (a).
 27. A process forpreparing a crystalline 1,2-ethanedisulfonic acid salt ofbiphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]-piperidin-4-ylester having a melting point greater than about 230° C., the processcomprising adding a seed crystal of a crystalline 1,2-ethanedisulfonicacid salt of biphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester to a solution comprising a 1,2-ethanedisulfonic acid salt ofbiphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester dissolved in an inert diluent, wherein the seed crystal has amelting point greater than about 230° C.
 28. A process for preparing acrystalline 1,2-ethanedisulfonic acid salt of biphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester having a melting point greater than about 230° C., the processcomprising: (a) dissolving a crystalline 1,2-ethanedisulfonic acid saltof biphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester in an inert diluent at a first temperature; (b) cooling theproduct of step (a) to a second temperature; and (c) adding a seedcrystal of 1,2-ethanedisulfonic acid salt of biphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester; wherein the seed crystal has a melting point higher than about230° C., the first temperature is a temperature sufficient to dissolvethe 1,2-ethanedisulfonic acid salt, and the second temperature is belowa temperature at which the seed crystal completely dissolves when addedto the product of step (b).
 29. A process for purifyingbiphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester; the process comprising forming a crystalline 1,2-ethanedisulfonicacid salt of biphenyl-2-ylcarbamic acid1-[2-(2-chloro-4-{[(R)-2-hydroxy-2-(8-hydroxy-2-oxo-1,2-dihydroquinolin-5-yl)ethylamino]methyl}-5-methoxyphenylcarbamoyl)ethyl]piperidin-4-ylester.